Evidence that the expression of transferrin receptor 1 on erythroid marrow cells mediates hepcidin suppression in the liver
2015; Elsevier BV; Volume: 43; Issue: 6 Linguagem: Inglês
10.1016/j.exphem.2015.03.001
ISSN1873-2399
AutoresSioḃán Keel, Raymond T. Doty, Li Liu, Elizabeta Nemeth, Sindhu Cherian, Tomas Ganz, Janis L. Abkowitz,
Tópico(s)Erythrocyte Function and Pathophysiology
Resumo•Transferrin receptor 1 expression on erythroid precursors may be a proximal mediator of the erythroid regulator of hepcidin expression. Hepcidin is the key regulator of iron absorption and recycling, and its expression is suppressed by red blood cell production. When erythropoiesis is expanded, hepcidin expression decreases. To gain insight into the stage of erythroid differentiation at which the regulation might originate, we measured serum hepcidin levels in archived pure red cell aplasia samples from patients whose block in erythroid differentiation was well defined by hematopoietic colony assays and marrow morphologic review. Hepcidin values are high or high normal in pure red cell aplasia patients in whom erythropoiesis is inhibited prior to the proerythroblast stage, but are suppressed in patients with excess proerythroblasts and few later erythroid cells. These data suggest that the suppressive effect of erythropoietic activity on hepcidin expression can arise from proerythroblasts, the stage at which transferrin receptor 1 expression peaks, prompting the hypothesis that transferrin receptor 1 expression on erythroid precursors is a proximal mediator of the erythroid regulator of hepcidin expression. Our characterization of erythropoiesis, iron status, and hepcidin expression in mice with global or hematopoietic cell-specific haploinsufficiency of transferrin receptor 1 provides initial supporting data for this model. The regulation appears independent of erythroferrone and growth differentiation factor 15, supporting the concept that several mechanisms signal iron need in response to an expanded erythron. Hepcidin is the key regulator of iron absorption and recycling, and its expression is suppressed by red blood cell production. When erythropoiesis is expanded, hepcidin expression decreases. To gain insight into the stage of erythroid differentiation at which the regulation might originate, we measured serum hepcidin levels in archived pure red cell aplasia samples from patients whose block in erythroid differentiation was well defined by hematopoietic colony assays and marrow morphologic review. Hepcidin values are high or high normal in pure red cell aplasia patients in whom erythropoiesis is inhibited prior to the proerythroblast stage, but are suppressed in patients with excess proerythroblasts and few later erythroid cells. These data suggest that the suppressive effect of erythropoietic activity on hepcidin expression can arise from proerythroblasts, the stage at which transferrin receptor 1 expression peaks, prompting the hypothesis that transferrin receptor 1 expression on erythroid precursors is a proximal mediator of the erythroid regulator of hepcidin expression. Our characterization of erythropoiesis, iron status, and hepcidin expression in mice with global or hematopoietic cell-specific haploinsufficiency of transferrin receptor 1 provides initial supporting data for this model. The regulation appears independent of erythroferrone and growth differentiation factor 15, supporting the concept that several mechanisms signal iron need in response to an expanded erythron. The human body requires ∼20–25 mg of iron per day to maintain its daily red cell production. The iron is provided mainly by macrophages that retrieve it from senescent red cells and, in small part, by intestinal iron absorption. Transferrin-bound iron in the blood is then delivered to developing erythroid precursors in the bone marrow, which require transferrin receptor 1 (TFRC) for adequate iron uptake [1Trenor III, C.C. Campagna D.R. Sellers V.M. Andrews N.C. Fleming M.D. The molecular defect in hypotransferrinemic mice.Blood. 2000; 96: 1113-1118Crossref PubMed Google Scholar, 2Levy J.E. Jin O. Fujiwara Y. Kuo F. Andrews N.C. Transferrin receptor is necessary for development of erythrocytes and the nervous system.Nat Genet. 1999; 21: 396-399Crossref PubMed Scopus (430) Google Scholar]. On the basis of ferrokinetic studies, Finch proposed that intestinal iron absorption and the mobilization of iron from stores in macrophages and hepatocytes are controlled by both a stores regulator and an erythroid regulator [3Finch C. Regulators of iron balance in humans.Blood. 1994; 84: 1697-1702Crossref PubMed Google Scholar]. The stores regulator is responsible for meeting the body's normal iron requirements and for maintaining iron stores, whereas the erythroid regulator ensures an adequate iron supply to the erythron, regardless of the body's iron balance. Hepcidin is key to iron metabolism because it is the common mediator of both the stores and erythroid regulators. Hepcidin acts by binding the iron export protein, ferroportin, leading to its degradation [4Nemeth E. Tuttle M.S. Powelson J. et al.Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization.Science. 2004; 306: 2090-2093Crossref PubMed Scopus (3439) Google Scholar]; this inhibits dietary iron absorption and macrophage iron recycling. Hepcidin synthesis is increased by an excess of iron and decreased by erythropoietic activity [5Hentze M.W. Muckenthaler M.U. Andrews N.C. Balancing acts: Molecular control of mammalian iron metabolism.Cell. 2004; 117: 285-297Abstract Full Text Full Text PDF PubMed Scopus (1347) Google Scholar, 6Pak M. Lopez M.A. Gabayan V. Ganz T. Rivera S. Suppression of hepcidin during anemia requires erythropoietic activity.Blood. 2006; 108: 3730-3735Crossref PubMed Scopus (379) Google Scholar, 7Vokurka M. Krijt J. Sulc K. Necas E. Hepcidin mRNA levels in mouse liver respond to inhibition of erythropoiesis.Physiol Res. 2006; 55: 667-674PubMed Google Scholar]. In some human diseases and in murine models (e.g., transferrin-deficient mice [8Bartnikas T.B. Andrews N.C. Fleming M.D. Transferrin is a major determinant of hepcidin expression in hypotransferrinemic mice.Blood. 2011; 117: 630-637Crossref PubMed Scopus (63) Google Scholar], β-thalassemia, and congenital dyserythropoietic anemia [9Kearney S.L. Nemeth E. Neufeld E.J. et al.Urinary hepcidin in congenital chronic anemias.Pediatr Blood Cancer. 2007; 48: 57-63Crossref PubMed Scopus (137) Google Scholar, 10Papanikolaou G. Tzilianos M. Christakis J.I. et al.Hepcidin in iron overload disorders.Blood. 2005; 105: 4103-4105Crossref PubMed Scopus (350) Google Scholar]), there is both iron overload and anemia, and thus coexisting signals to both up-regulate and down-regulate hepcidin expression. In these conditions, the erythroid regulator is dominant. Exactly how an erythroid precursor in the bone marrow communicates its iron need to hepatocytes remains unknown. Data suggest that the regulator is a soluble molecule [11Weizer-Stern O. Adamsky K. Amariglio N. et al.Downregulation of hepcidin and haemojuvelin expression in the hepatocyte cell-line HepG2 induced by thalassaemic sera.Br J Haematol. 2006; 135: 129-138Crossref PubMed Scopus (60) Google Scholar]. The erythrokine growth differentiation factor 15 (GDF-15), which is markedly elevated in β-thalassemic serum, was identified as one possible regulator in pathologic states [12Tanno T. Bhanu N.V. Oneal P.A. et al.High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin.Nat Med. 2007; 13: 1096-1101Crossref PubMed Scopus (644) Google Scholar], though some data refute this [13Fertrin K.Y. Lanaro C. Franco-Penteado C.F. et al.Erythropoiesis-driven regulation of hepcidin in human red cell disorders is better reflected through concentrations of soluble transferrin receptor rather than growth differentiation factor 15.Am J Hematol. 2014; 89: 385-390Crossref PubMed Scopus (19) Google Scholar]. Accumulating data suggest that GDF-15 is unlikely to mediate hepcidin suppression in normal erythropoiesis and during acute erythropoietic stress [14Kanda J. Mizumoto C. Kawabata H. et al.Serum hepcidin level and erythropoietic activity after hematopoietic stem cell transplantation.Haematologica. 2008; 93: 1550-1554Crossref PubMed Scopus (48) Google Scholar, 15Ashby D.R. Gale D.P. Busbridge M. et al.Erythropoietin administration in humans causes a marked and prolonged reduction in circulating hepcidin.Haematologica. 2010; 95: 505-508Crossref PubMed Scopus (142) Google Scholar, 16Tanno T. Rabel A. Lee Y.T. Yau Y.Y. Leitman S.F. Miller J.L. Expression of growth differentiation factor 15 is not elevated in individuals with iron deficiency secondary to volunteer blood donation.Transfusion. 2010; 50: 1532-1535Crossref PubMed Scopus (29) Google Scholar]. Although the recently characterized erythroid factor erythroferrone (Erfe) is capable of suppressing hepcidin expression under erythropoietic stress [17Kautz L. Jung G. Valore E.V. Rivella S. Nemeth E. Ganz T. Identification of erythroferrone as an erythroid regulator of iron metabolism.Nat Genet. 2014; 46: 678-684Crossref PubMed Scopus (679) Google Scholar], its role in homeostasis is unknown. As the regulator originates from the erythroid marrow [6Pak M. Lopez M.A. Gabayan V. Ganz T. Rivera S. Suppression of hepcidin during anemia requires erythropoietic activity.Blood. 2006; 108: 3730-3735Crossref PubMed Scopus (379) Google Scholar, 7Vokurka M. Krijt J. Sulc K. Necas E. Hepcidin mRNA levels in mouse liver respond to inhibition of erythropoiesis.Physiol Res. 2006; 55: 667-674PubMed Google Scholar], we studied an instructive group of patients with pure red cell aplasia (PRCA) to determine which stages of erythropoiesis signal hepcidin suppression. PRCA is characterized by severe normochromic, normocytic, or macrocytic anemia associated with reticulocytopenia and the near absence of hemoglobin-containing cells in an otherwise normal marrow aspirate. Therefore, there is maturation arrest at or before the proerythroblast stage [18Keel S. Abkowitz J. Pure red cell aplasia.in: Young N.S. Gerson S.L. High K.A. Clinical Hematology. Maryland Heights. Elsevier (Mosby), Mo2006Google Scholar]. In the study described here, we measured hepcidin levels in stored serum from a cohort of immunologically mediated PRCA patients whose block in erythroid differentiation was previously established [19Charles R.J. Sabo K.M. Kidd P.G. Abkowitz J.L. The pathophysiology of pure red cell aplasia: Implications for therapy.Blood. 1996; 87: 4831-4838PubMed Google Scholar]. Our data suggest that the erythroid regulator of hepcidin expression can derive from proerythroblasts, but not from less-differentiated erythroid progenitors. Recognizing that TFRC expression peaks on proerythroblasts [20Iacopetta B.J. Morgan E.H. Yeoh G.C. Transferrin receptors and iron uptake during erythroid cell development.Biochim Biophys Acta. 1982; 687: 204-210Crossref PubMed Scopus (126) Google Scholar] and after considering the published data regarding hepcidin regulation in murine studies and human disorders, we hypothesized that TFRC is a proximal mediator of the erythroid regulator of hepcidin expression and tested this directly in a murine model of Tfrc haploinsufficiency. Sera, obtained from patients with PRCA who were evaluated at the University of Washington between 1982 and 1992, were stored at −80°C. Patient characteristics were previously published [19Charles R.J. Sabo K.M. Kidd P.G. Abkowitz J.L. The pathophysiology of pure red cell aplasia: Implications for therapy.Blood. 1996; 87: 4831-4838PubMed Google Scholar]. Hepcidin [21Ganz T. Olbina G. Girelli D. Nemeth E. Westerman M. Immunoassay for human serum hepcidin.Blood. 2008; 112: 4292-4297Crossref PubMed Scopus (547) Google Scholar] and GDF-15 assays (Quantikine Human GDF-15 Immunoassay, R&D Systems, Minneapolis, MN, USA) were performed blinded to diagnoses. Hepcidin concentration in a recent PRCA case was similar to those in the archived samples at the same stage of erythroid differentiation block, indicating that hepcidin in archived samples did not significantly degrade. S.C. rereviewed the marrow morphology in available specimens. The University of Washington Human Subjects Committee approved all studies. Mice with haploinsufficiency of Tfrc were obtained from Nancy Andrews (Duke School of Medicine) on a C57BL/6 background [2Levy J.E. Jin O. Fujiwara Y. Kuo F. Andrews N.C. Transferrin receptor is necessary for development of erythrocytes and the nervous system.Nat Genet. 1999; 21: 396-399Crossref PubMed Scopus (430) Google Scholar] and housed at University of Washington. Polymerase chain reaction (PCR) genotyping was performed on tail biopsy DNA (list of primers is provided in Supplementary Table E1, online only, available at www.exphem.org). Ten million bone marrow cells were transplanted into 6- to 8-week-old Pep3b lethally irradiated recipients. Mice were sacrificed for analyses after stable engraftment and at least 7 weeks posttransplantation. The University of Washington Institutional Animal Care and Use Committee approved all studies. Mice were given 100 U epoetin alfa (Centocor Ortho Biotech, Horsham, PA, USA) intraperitoneally and sacrificed 15 hours later. Linear regression analysis was performed using GraphPad Prism Version 5.0 (GraphPad Software, La Jolla, CA, USA). Mice were bled retro-orbitally. Reticulocyte counts were obtained at Phoenix Laboratory (Everett, WA, USA) on an ADVIA120 (Siemens Medical Solutions USA, Malvern, PA). Complete blood counts were obtained on a Hemavet HV950FS (Drew Scientific, Waterbury, CT) analyzer. Erythropoietin was measured using Mouse/Rat EPO Immunoassay Kit (R&D Systems). Serum iron parameters were determined using an iron/TIBC kit (Pointe Scientific, Canton, MI, USA). Nonheme tissue iron content was quantified as described [22Torrance J.D. Bothwell T.H. Tissue iron stores.in: Cook J.D. Methods in Hematology: Iron. Churchill Livingstone, New York1980: 90-115Google Scholar]. Soluble transferrin receptor was measured by an enzyme-linked immunosorbent assay (MyBioSource, San Diego, CA, USA). Mice were sacrificed by cervical dislocation under 2,2,2-tribromoethanol. Total RNA was isolated using Trizol (Life Technologies, Grand Island, NY, USA). Complementary DNA was synthesized using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA), and murine liver hepcidin, Bmp6, and Id1 messenger RNA (mRNA) levels were determined with 5′-nuclease quantitative PCR assays. Gene expression was quantified by the Pfaffl method [23Pfaffl M.W. A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (24283) Google Scholar] using the mean of duplicate samples relative to β-actin expression. Expression of Erfe in FACSAria-sorted hematopoietic cell populations with the FACSAria (BD Biosciences, San Jose, CA, USA) was measured by SYBR green quantitative PCR assays and normalized to β-actin expression (primers in Supplementary Table E1, online only, available at www.exphem.org). Single-cell suspensions of freshly isolated marrow or spleen were immunostained with antibodies to Ter119, CD44, CD71, B220, and Gr1 (BD Pharmingen, Franklin Lakes, NJ, USA) [24Chen K. Liu J. Heck S. Chasis J.A. An X. Mohandas N. Resolving the distinct stages in erythroid differentiation based on dynamic changes in membrane protein expression during erythropoiesis.Proc Natl Acad Sci U S A. 2009; 106: 17413-17418Crossref PubMed Scopus (317) Google Scholar, 25Socolovsky M. Nam H.S. Fleming M.D. Haase V.H. Brugnara C. Lodish H.F. Ineffective erythropoiesis in Stat5a–/–5b–/– mice due to decreased survival of early erythroblasts.Blood. 2001; 98: 3261-3273Crossref PubMed Scopus (562) Google Scholar]. B220+Gr1+ cells were excluded from analyses. Additional cells were fixed with 1% paraformaldehyde, permeabilized with ice-cold methanol, and immunostained with an antibody recognizing phospho-STAT5(Y694). Staining was quantified with a FACSCanto Flow Cytometer (BD Biosciences), and data were analyzed with FlowJo Tree Star (FlowJo, Ashland, OR, USA). The geometric mean fluorescence of CD71 (Tfrc) expression in each population was normalized to the wild-type average of population I/II, which was defined as 100%. Absolute marrow cellularity was calculated according to Colvin et al. [26Colvin G.A. Lambert J.F. Abedi M. et al.Murine marrow cellularity and the concept of stem cell competition: Geographic and quantitative determinants in stem cell biology.Leukemia. 2004; 18: 575-583Crossref PubMed Scopus (76) Google Scholar]. We defined erythroid Tfrc mass as the product of the absolute number of cells in a gated population and the normalized CD71 expression of that population, calculated separately for marrow and spleen populations, and then added together to obtain the absolute Tfrc mass of each population. To minimize the confounding effects of transfusion or immunosuppressive therapy on hepcidin expression, we restricted our analysis to those PRCA patients evaluated at disease presentation (serum samples were available from 13 of the 21 patients in the original cohort who met this criterion). Clinical data were previously published (summarized in Supplementary Table E2, online only, available at www.exphem.org). We defined four stages of differentiation blocks: before BFU-E (burst-forming units—erythroid, i.e., no colony growth), BFU-E to CFU-E (colony-forming units—erythroid, i.e., BFU-E are present, but CFU-E are not detected), CFU-E to proerythroblast (i.e., BFU-E and CFU-E are present, but proerythroblasts are rare or absent), and proerythroblast stage (i.e., ample to excess proerythroblasts). In the current study, we repeated a pathologic review of seven available marrow aspirates and one newly diagnosed PRCA patient's aspirate to confirm the diagnosis and enumerate the number of proerythroblasts present relative to total marrow erythroid mononuclear cells. We confirmed that there was a relative increase in proerythroblasts in two marrows (patients 24 and 2). The ratios of proerythroblasts to total marrow erythroid mononuclear cells were 0.82, and 0.77, respectively, whereas the ratio ranged from 0 to 0.50 in the other evaluable patient samples. All 11 patients in whom erythropoiesis was blocked at or prior to CFU-E had high normal (3/11) or elevated (8/11) hepcidin levels (mean hepcidin level was 490.2 ± 110.2 ng/mL compared with 42.0 ± 22.3 ng/mL in control samples, p = 0.002) (Supplementary Figure E1A, online only, available at www.exphem.org). In contrast, hepcidin was suppressed in one of the two patients in whom erythroid differentiation was blocked at the proerythroblast stage (patient 2, 12.7 ng/mL). Importantly, patient 2 had the highest number of proerythroblasts relative to total marrow mononuclear cells among all PRCA patients analyzed (0.21 vs. 0–0.08) (Supplementary Figure E1B, online only, available at www.exphem.org). Although patient 24 also had a near-complete block in erythroid differentiation at the proerythroblast stage, his hepcidin was not suppressed; of note, he had many fewer proerythroblasts (ratio = 0.08). As GDF-15 is implicated in the inhibition of hepcidin in pathologic conditions [12Tanno T. Bhanu N.V. Oneal P.A. et al.High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin.Nat Med. 2007; 13: 1096-1101Crossref PubMed Scopus (644) Google Scholar], we also measured serum GDF-15 levels; these values did not correlate with the site of block or clinical parameters (Supplementary Figure E2, online only, available at www.exphem.org). These data imply that proerythroblasts can signal the liver to suppress hepcidin expression, but do not include or exclude the possibility that later erythroid cells can also signal. As TFRC expression increases at the CFU-E stage and peaks on proerythroblasts before decreasing [20Iacopetta B.J. Morgan E.H. Yeoh G.C. Transferrin receptors and iron uptake during erythroid cell development.Biochim Biophys Acta. 1982; 687: 204-210Crossref PubMed Scopus (126) Google Scholar], we considered whether the abundance of TFRC protein might be a physiologic way that the erythron judges its need for iron. We reasoned that patient 2 had excess proerythroblasts, each with high TFRC expression, and thus a relatively high total TFRC mass that might signal hepcidin suppression. Patients with a block in erythroid differentiation prior to proerythroblasts would have low TFRC protein on all marrow cells and would be unable to signal hepcidin suppression. Recognizing that our hepcidin data have multiple confounding variables (e.g., hepcidin values are known to vary with age, sex, transfusion, time of day, menopausal status, and potential hepcidin degradation caused by prolonged storage of serum samples) and that the hypothesis resulted from an observation in a single patient, we tested the relationship of erythroid cell surface TFRC levels to hepcidin expression directly and quantitatively in a murine model. Tfrc+/– mice have iron-restricted erythropoiesis characterized by a mild microcytic, hypochromic anemia (Table 1) and a relative and absolute expansion of early marrow erythroid precursors (population III, which represents polychromatophilic erythroblasts [27Liu S. Bhattacharya S. Han A. et al.Haem-regulated eIF2alpha kinase is necessary for adaptive gene expression in erythroid precursors under the stress of iron deficiency.Br J Haematol. 2008; 143: 129-137Crossref PubMed Scopus (37) Google Scholar], 16.8 ± 2.2% vs. 13.0 ± 1.2%, p = 0.002, and 4.9 ± 1.0 × 107 vs. 3.5 ± 0.4 × 107, p = 0.004) (Fig. 1). Splenic analyses did not reveal significant differences in relative or absolute numbers of erythroid precursors as they were not consistently expanded in Tfrc+/− mice, confirming that the animals model physiologic and not stress erythropoiesis (Supplementary Figure E3, online only, available at www.exphem.org). Thus, Tfrc+/− mice have iron-restricted and mildly expanded erythropoiesis, consistent with previous reports [2Levy J.E. Jin O. Fujiwara Y. Kuo F. Andrews N.C. Transferrin receptor is necessary for development of erythrocytes and the nervous system.Nat Genet. 1999; 21: 396-399Crossref PubMed Scopus (430) Google Scholar].Table 1Hematological parameters in 12-week old Tfrc+/− and control miceTfrc+/+ (n = 7)Tfrc+/− (n = 7)p ValueHemoglobin (g/dL)14.6 ± 0.513.8 ± 0.30.003Hematocrit (%)53.4 ± 7.646.8 ± 5.4NSRed blood cells (M/μL)10.4 ± 0.611.5 ± 0.30.002Mean corpuscular volume (fL)51.2 ± 4.840.8 ± 3.90.0008Mean corpuscular hemoglobin (pg)14.1 ± 0.512.1 ± 0.4<0.0001Reticulocytes (K/μL)355.1 ± 64.0451.3 ± 41.80.006Erythropoietin (pg/mL)491.7 ± 392.8326.3 ± 164.5NSPlatelets (K/μL)713.6 ± 265.7764.9 ± 117.6NSWhite blood cells (K/μL)11.0 ± 4.110.2 ± 4.9NSNS = not significant. Open table in a new tab NS = not significant. As expected, Tfrc levels per cell are decreased on Tfrc+/− compared with control cells at all stages of terminal erythroid differentiation (Fig. 2). Additionally, the total quantity of cell surface transferrin receptor present on marrow and splenic erythroid precursors calculated as a function of mean fluorescence intensity and cell number (and termed erythroid Tfrc mass) is lower on the Tfrc+/− erythron compared with controls despite the early erythroid expansion (11.7 ± 2.4 × 107 vs. 7.9 ± 1.1 × 107, p = 0.003). As a secondary study, we measured soluble Tfrc concentration in serum [13Fertrin K.Y. Lanaro C. Franco-Penteado C.F. et al.Erythropoiesis-driven regulation of hepcidin in human red cell disorders is better reflected through concentrations of soluble transferrin receptor rather than growth differentiation factor 15.Am J Hematol. 2014; 89: 385-390Crossref PubMed Scopus (19) Google Scholar, 28Huebers H.A. Beguin Y. Pootrakul P. Einspahr D. Finch C.A. Intact transferrin receptors in human plasma and their relation to erythropoiesis.Blood. 1990; 75: 102-107Crossref PubMed Google Scholar]. Soluble Tfrc concentration was equivalent in Tfrc+/− and controls (Table 2); we suspect this is a consequence of the higher absolute numbers of polychromatophilic erythroblasts and reticulocyte counts observed in Tfrc+/− compared with control animals, as these populations are known to preferentially contribute to soluble Tfrc levels in vivo [13Fertrin K.Y. Lanaro C. Franco-Penteado C.F. et al.Erythropoiesis-driven regulation of hepcidin in human red cell disorders is better reflected through concentrations of soluble transferrin receptor rather than growth differentiation factor 15.Am J Hematol. 2014; 89: 385-390Crossref PubMed Scopus (19) Google Scholar, 29R'Zik S. Loo M. Beguin Y. Reticulocyte transferrin receptor (TfR) expression and contribution to soluble TfR levels.Haematologica. 2001; 86: 244-251PubMed Google Scholar]. Splenic erythropoiesis, which is comparable in Tfrc+/− and control mice, constitutes only ∼10% of the total steady-state erythroid compartment and, thus, contributes little to the total erythroid Tfrc mass (Supplementary Figure E3, online only, available at www.exphem.org).Table 2Iron parameters in 11- to 15-week-old male Tfrc+/− and control miceTfrc+/+ (n = 8)Tfrc+/− (n = 10)p ValueSerum iron (μg/dL)95.5 ± 15.984.2 ± 10.20.08Transferrin saturation (%)28.8 ± 4.227.1 ± 4.80.45Liver iron (μg/g)56.1 ± 9.642.5 ± 8.60.01Kidney iron (μg/g)44.2 ± 3.638.8 ± 4.30.01Spleen iron μg/g400.6 ± 96.7284.7 ± 99.10.02 μg25.0 ± 5.918.0 ± 5.90.02Soluble transferrin receptor (ng/mL)5.1 ± 2.64.4 ± 2.50.57Erythropoietin (pg/mL)504 ± 372514 ± 3100.95 Open table in a new tab Humans and mice with iron-restrictive erythropoiesis caused by dietary iron deficiency [21Ganz T. Olbina G. Girelli D. Nemeth E. Westerman M. Immunoassay for human serum hepcidin.Blood. 2008; 112: 4292-4297Crossref PubMed Scopus (547) Google Scholar] or a lack of transferrin [8Bartnikas T.B. Andrews N.C. Fleming M.D. Transferrin is a major determinant of hepcidin expression in hypotransferrinemic mice.Blood. 2011; 117: 630-637Crossref PubMed Scopus (63) Google Scholar] have early erythroid expansion and suppressed hepcidin [27Liu S. Bhattacharya S. Han A. et al.Haem-regulated eIF2alpha kinase is necessary for adaptive gene expression in erythroid precursors under the stress of iron deficiency.Br J Haematol. 2008; 143: 129-137Crossref PubMed Scopus (37) Google Scholar]. Because Tfrc+/− mice have iron-restricted-erythropoiesis and also early erythroid expansion, we expected hepcidin to be lower in Tfrc+/− mice than in controls. However, Tfrc+/− mice had hepcidin expression comparable to that of control animals (Fig. 3A). Importantly, serum transferrin saturations were comparable to those of control mice, whereas liver iron content was lower in Tfrc+/− than in control mice, so any contribution of the stores regulator (which stimulates hepcidin expression) to hepcidin expression in Tfrc+/− mice should be equivalent to or less than that of controls (Table 2). Consistent with equivalent serum iron availability, expression of bone morphogenetic protein 6 (Bmp6) and inhibitor of DNA binding 1 (Id1) [30Kautz L. Meynard D. Monnier A. et al.Iron regulates phosphorylation of Smad1/5/8 and gene expression of Bmp6, Smad7, Id1, and Atoh8 in the mouse liver.Blood. 2008; 112: 1503-1509Crossref PubMed Scopus (335) Google Scholar] were not reduced in Tfrc+/− mice (Supplementary Figure E4, online only, available at www.exphem.org). These data imply that the erythroid regulator of hepcidin suppression is not active in Tfrc+/− mice and are consistent with our working model that Tfrc is needed to suppress hepcidin expression. The liver is the physiologically important site for modulating hepcidin expression in response to body iron status. Specifically, it is proposed that Hfe−Tfrc complexes on the surface of hepatocytes sense the saturation of iron-bound transferrin in the serum; at low transferrin saturations, Hfe is sequestered by Tfrc, and as transferrin saturation increases, Hfe is dislodged from its overlapping binding site on Tfrc by holotransferrin and is then free to interact with transferrin receptor 2 and signal the upregulation of hepcidin expression [31Schmidt P.J. Toran P.T. Giannetti A.M. Bjorkman P.J. Andrews N.C. The transferrin receptor modulates Hfe-dependent regulation of hepcidin expression.Cell Metab. 2008; 7: 205-214Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar]. To exclude any confounding liver-specific effect of Tfrc haploinsufficiency on hepcidin expression and, thus, on interpretation of data centered on the erythroid regulator of hepcidin specifically, we transplanted Tfrc+/− (or control) marrow into lethally irradiated wild-type recipients and confirmed that, analogous to Tfrc+/− mice, mice with hematopoietic cell-specific Tfrc haploinsufficiency also have iron-restricted erythropoiesis (Table 3), reduced Tfrc levels per cell at all stages of terminal erythroid differentiation (Fig. 4), and reduced erythroid Tfrc mass (3.3 ± 0.6 × 107 vs. 7.1 ± 2.4 × 107, n = 3 mice per group, p = 0.058), and percentage transferrin saturation is not significantly reduced compared with that of control mice (Table 4). Hepcidin levels are higher in these mice than in controls, and this difference is significant among male mice (3.9 ± 1.4 vs. 1.0 ± 0.6, p = 0.03) (Fig. 3B). Thus, the lack of hepcidin suppression in the face of iron-restricted erythropoiesis observed in Tfrc+/− mice holds in the transplant model.Table 3Hematologic parameters in mice transplanted with control or Tfrc+/− marrow 7–8 weeks after transplantTfrc+/+ (n = 10)Tfrc+/− (n = 10)p ValueHemoglobin (g/dL)14.6 ± 0.413.7 ± 1.00.02Hematocrit (%)47.0 ± 1.243.3 ± 2.2<0.001Red blood cells (M/μL)10.1 ± 0.311.5 ± 0.6<0.001Mean corpuscular volume (fL)46.7 ± 0.437.8 ± 0.9<0.001Mean corpuscular hemoglobin (pg)14.5 ± 0.212.0 ± 0.4<0.001Reticulocytes (K/μL)337 ± 25400 ± 470.11 (n = 3)Erythropoietin (pg/mL)1033 ± 3181676 ± 4410.11 (n = 3)Platelets (K/μL)881 ± 100885 ± 1450.95White blood cells (K/μL)16.4 ± 3.616.4 ± 3.51.0 Open table in
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