Tmprss6-mutated mask mice display iron deficiency anemia and high expression of hepcidin. The aim of the study was to determine the effect of erythropoietin administration on proteins participating in the control of iron homeostasis in the liver and spleen in C57BL/6 and mask mice. Administration of erythropoietin for four days at 50 IU/mouse/day increased hemoglobin and hematocrit in C57BL/6 mice, no such increase was seen in mask mice. Erythropoietin administration decreased hepcidin expression in C57BL/6 mice, but not in mask mice. Erythropoietin treatment significantly increased the spleen size in both C57BL/6 and mask mice. Furthermore, erythropoietin administration increased splenic Fam132b, Fam132a and Tfr2 mRNA content. At the protein level, erythropoietin increased the amount of splenic erythroferrone and transferrin receptor 2 both in C57BL/6 and mask mice. Splenic ferroportin content was decreased in erythropoietin-treated mask mice in comparison with erythropoietin-treated C57BL/6 mice. In mask mice, the amount of liver hemojuvelin was decreased in comparison with C57BL/6 mice. The pattern of hemojuvelin cleavage was different between C57BL/6 and mask mice: In both groups, a main hemojuvelin band was detected at approximately 52 kDa; in C57BL/6 mice, a minor cleaved band was seen at 47 kDa. In mask mice, the 47 kDa band was absent, but additional minor bands were detected at approximately 45 kDa and 48 kDa. The results provide support for the interaction between TMPRSS6 and hemojuvelin in vivo; they also suggest that hemojuvelin could be cleaved by another as yet unknown protease in the absence of functional TMPRSS6. The lack of effect of erythropoietin on hepcidin expression in mask mice can not be explained by changes in erythroferrone synthesis, as splenic erythroferrone content increased after erythropoietin administration in both C57BL/6 and mask mice.

Faculty of Pharmacy in Hradec Králové Charles University Hradec Králové Czech Republic

Institute of Pathological Physiology 1st Faculty of Medicine Charles University Prague Czech Republic

Laboratory of Tumour Resistance Institute of Biotechnology BIOCEV Research Center Czech Academy of Sciences Vestec Czech Republic

Zobrazit více v PubMed

Pasricha SR, Drakesmith H. Iron Deficiency Anemia: Problems in Diagnosis and Prevention at the Population Level. Hematol Oncol Clin North Am. 2016;30: 309–25. doi: 10.1016/j.hoc.2015.11.003 PubMed DOI

Finberg KE, Heeney MM, Campagna DR, Aydinok Y, Pearson HA, Hartman KR, et al. Mutations in TMPRSS6 cause iron-refractory iron deficiency anemia (IRIDA). Nat Genet. 2008;40: 569–71. doi: 10.1038/ng.130 PubMed DOI PMC

Du X, She E, Gelbart T, Truksa J, Lee P, Xia Y, et al. The serine protease TMPRSS6 is required to sense iron deficiency. Science 2008;320: 1088–92. doi: 10.1126/science.1157121 PubMed DOI PMC

Folgueras AR, de Lara FM, Pendás AM, Garabaya C, Rodríguez F, Astudillo A, et al. Membrane-bound serine protease matriptase-2 (TMPRSS6) is an essential regulator of iron homeostasis. Blood. 2008;112: 2539–45. doi: 10.1182/blood-2008-04-149773 PubMed DOI

Guillem F, Lawson S, Kannengiesser C, Westerman M, Beaumont C, Grandchamp B. Two nonsense mutations in the TMPRSS6 gene in a patient with microcytic anemia and iron deficiency. Blood. 2008;112: 2089–91. doi: 10.1182/blood-2008-05-154740 PubMed DOI

Melis MA, Cau M, Congiu R, Sole G, Barella S, Cao A, et al. A mutation in the TMPRSS6 gene, encoding a transmembrane serine protease that suppresses hepcidin production, in familial iron deficiency anemia refractory to oral iron. Haematologica. 2008;93: 1473–9. doi: 10.3324/haematol.13342 PubMed DOI

Pigeon C, Ilyin G, Courselaud B, Leroyer P, Turlin B, Brissot P, et al. A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem. 2001;276: 7811–9. doi: 10.1074/jbc.M008923200 PubMed DOI

Nicolas G, Bennoun M, Devaux I, Beaumont C, Grandchamp B, Kahn A, et al. Lack of hepcidin gene expression and severe tissue iron overload in upstream stimulatory factor 2 (USF2) knockout mice. Proc Natl Acad Sci U S A. 2001;98: 8780–5. doi: 10.1073/pnas.151179498 PubMed DOI PMC

Nicolas G, Bennoun M, Porteu A, Mativet S, Beaumont C, Grandchamp B, et al. Severe iron deficiency anemia in transgenic mice expressing liver hepcidin. Proc Natl Acad Sci U S A. 2002;99: 4596–601. doi: 10.1073/pnas.072632499 PubMed DOI PMC

Roetto A, Papanikolaou G, Politou M, Alberti F, Girelli D, Christakis J, et al. Mutant antimicrobial peptide hepcidin is associated with severe juvenile hemochromatosis. Nat Genet. 2003;33: 21–2. doi: 10.1038/ng1053 PubMed DOI

Nemeth E, Tuttle MS, Powelson J, Vaughn MB, Donovan A, Ward DM, et al. Hepcidin regulates cellular iron efflux by binding to ferroportin and inducing its internalization. Science. 2004;306: 2090–3. doi: 10.1126/science.1104742 PubMed DOI

Frazer DM, Wilkins SJ, Mirciov CS, Dunn LA, Anderson GJ. Hepcidin independent iron recycling in a mouse model of β-thalassaemia intermedia. Br J Haematol. 2016;175: 308–317. doi: 10.1111/bjh.14206 PubMed DOI

Nicolas G, Viatte L, Bennoun M, Beaumont C, Kahn A, Vaulont S. Hepcidin, a new iron regulatory peptide. Blood Cells Mol Dis. 2002;29: 327–35. PubMed

Vokurka M, Krijt J, Sulc K, Necas E. Hepcidin mRNA levels in mouse liver respond to inhibition of erythropoiesis. Physiol Res. 2006;55: 667–74. PubMed

Kautz L, Jung G, Valore EV, Rivella S, Nemeth E, Ganz T. Identification of erythroferrone as an erythroid regulator of iron metabolism. Nat Genet. 2014;46: 678–84. doi: 10.1038/ng.2996 PubMed DOI PMC

Kim A, Nemeth E. New insights into iron regulation and erythropoiesis. Curr Opin Hematol. 2015;22: 199–205. doi: 10.1097/MOH.0000000000000132 PubMed DOI PMC

Lehmberg K, Grosse R, Muckenthaler MU, Altamura S, Nielsen P, Schmid H, et al. Administration of recombinant erythropoietin alone does not improve the phenotype in iron refractory iron deficiency anemia patients. Ann Hematol. 2013;92: 387–94. doi: 10.1007/s00277-012-1618-8 PubMed DOI

Nicolas G, Deschemin JC, Ramsay AJ, Mayeux P, Grandchamp B, Beaumont C, et al. Is EPO therapy able to correct iron deficiency anaemia caused by matriptase-2 deficiency? Br J Haematol. 2011;152: 498–500. doi: 10.1111/j.1365-2141.2010.08473.x PubMed DOI

Lee P. EPO-mediated reduction in Hamp expression in vivo corrects iron deficiency anaemia in TMPRSS6 deficiency—Erratum. Br J Haematol. 2012;156: 415–6. PubMed PMC

Nai A, Rubio A, Campanella A, Gourbeyre O, Artuso I, Bordini J, et al. Limiting hepatic Bmp-Smad signaling by matriptase-2 is required for erythropoietin-mediated hepcidin suppression in mice. Blood. 2016;127: 2327–36. doi: 10.1182/blood-2015-11-681494 PubMed DOI PMC

Silvestri L, Pagani A, Nai A, De Domenico I, Kaplan J, Camaschella C. The serine protease matriptase-2 (TMPRSS6) inhibits hepcidin activation by cleaving membrane hemojuvelin. Cell Metab. 2008;8: 502–11. doi: 10.1016/j.cmet.2008.09.012 PubMed DOI PMC

Niederkofler V, Salie R, Arber S. Hemojuvelin is essential for dietary ironsensing, and its mutation leads to severe iron overload. J Clin Invest. 2005;115: 2180–6. doi: 10.1172/JCI25683 PubMed DOI PMC

Huang FW, Pinkus JL, Pinkus GS, Fleming MD, Andrews NC. A mouse model of juvenile hemochromatosis. J Clin Invest. 2005;115: 2187–91. doi: 10.1172/JCI25049 PubMed DOI PMC

Finberg KE, Whittlesey RL, Fleming MD, Andrews NC. Down-regulation of Bmp/Smad signaling by Tmprss6 is required for maintenance of systemic iron homeostasis. Blood. 2010;115: 3817–26. doi: 10.1182/blood-2009-05-224808 PubMed DOI PMC

Nai A, Pagani A, Mandelli G, Lidonnici MR, Silvestri L, Ferrari G, et al. Deletion of TMPRSS6 attenuates the phenotype in a mouse model of β-thalassemia. Blood. 2012;119: 5021–9. doi: 10.1182/blood-2012-01-401885 PubMed DOI PMC

Frýdlová J, Fujikura Y, Vokurka M, Nečas E, Krijt J. Decreased hemojuvelin protein levels in mask mice lacking matriptase-2-dependent proteolytic activity. Physiol Res. 2013;62: 405–11. PubMed

Willemetz A, Lenoir A, Deschemin JC, Lopez-Otin C, Ramsay AJ, Vaulont S, et al. Matriptase-2 is essential for hepcidin repression during fetal life and postnatal development in mice to maintain iron homeostasis. Blood. 2014;124: 441–4. doi: 10.1182/blood-2014-01-551150 PubMed DOI

Aschemeyer S, Gabayan V, Ganz T, Nemeth E, Kautz L. Erythroferrone and matriptase-2 independently regulate hepcidin expression. Am J Hematol. 2017;92: E61–E63. doi: 10.1002/ajh.24672 PubMed DOI PMC

Frýdlová J, Přikryl P, Truksa J, Falke LL, Du X, Gurieva I, et al. Effect of erythropoietin, iron deficiency and iron overload on liver matriptase-2 (TMPRSS6) protein content in mice and rats. PLoS One. 2016. February4;11(2):e0148540 doi: 10.1371/journal.pone.0148540 PubMed DOI PMC

Gurieva I, Frýdlová J, Rychtarčíková Z, Vokurka M, Truksa J, Krijt J. Erythropoietin administration increases splenic erythroferrone protein content and liver TMPRSS6 protein content in rats. Blood Cells Mol Dis. 2017;64: 1–7. doi: 10.1016/j.bcmd.2017.02.007 PubMed DOI

Torrance JD, Bothwell TH. Tissue iron stores In: Cook JD, editor. Methods in hematology, Vol. 1 New York: Churchill Livingstone; 1981. p. 90–115.

Wallace DF, Secondes ES, Rishi G, Ostini L, McDonald CJ, Lane SW, et al. A critical role for murine transferrin receptor 2 in erythropoiesis during iron restriction. Br J Haematol. 2015;168: 891–901. doi: 10.1111/bjh.13225 PubMed DOI

Nai A, Lidonnici MR, Rausa M, Mandelli G, Pagani A, Silvestri L, et al. The second transferrin receptor regulates red blood cell production in mice. Blood. 2015;125: 1170–9. doi: 10.1182/blood-2014-08-596254 PubMed DOI PMC

Rishi G, Secondes ES, Wallace DF, Subramaniam VN. Hematopoietic deletion of transferrin receptor 2 in mice leads to a block in erythroid differentiation during iron-deficient anemia. Am J Hematol. 2016;91(8): 812–8. doi: 10.1002/ajh.24417 PubMed DOI

Canonne-Hergaux F, Donovan A, Delaby C, Wang HJ, Gros P. Comparative studies of duodenal and macrophage ferroportin proteins. Am J Physiol Gastrointest Liver Physiol. 2006;290: G156–63. doi: 10.1152/ajpgi.00227.2005 PubMed DOI

Knutson MD, Vafa MR, Haile DJ, Wessling-Resnick M. Iron loading and erythrophagocytosis increase ferroportin 1 (FPN1) expression in J774 macrophages. Blood. 2003;102: 4191–7. doi: 10.1182/blood-2003-04-1250 PubMed DOI

Paulson RF, Shi L, Wu DC. Stress erythropoiesis: new signals and new stress progenitor cells. Curr Opin Hematol. 2011;18: 139–45. doi: 10.1097/MOH.0b013e32834521c8 PubMed DOI PMC

Kim TS, Hanak M, Trampont PC, Braciale TJ. Stress-associated erythropoiesis initiation is regulated by type 1 conventional dendritic cells. J Clin Invest. 2015;125: 3965–80. doi: 10.1172/JCI81919 PubMed DOI PMC

Peng H, Truksa J, Lee P. EPO-mediated reduction in Hamp expression in vivo corrects iron deficiency anaemia in TMPRSS6 deficiency. Br J Haematol. 2010;151: 106–9. doi: 10.1111/j.1365-2141.2010.08306.x PubMed DOI PMC

Kautz L, Meynard D, Monnier A, Darnaud V, Bouvet R, Wang RH, 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: 1503–9. doi: 10.1182/blood-2008-03-143354 PubMed DOI

Seldin MM, Peterson JM, Byerly MS, Wei Z, Wong GW. Myonectin (CTRP15), a novel myokine that links skeletal muscle to systemic lipid homeostasis. J Biol Chem. 2012;287: 11968–80. doi: 10.1074/jbc.M111.336834 PubMed DOI PMC

Forejtníková H, Vieillevoye M, Zermati Y, Lambert M, Pellegrino RM, Guihard S, et al. Transferrin receptor 2 is a component of the erythropoietin receptor complex and is required for efficient erythropoiesis. Blood. 2010;116: 5357–67. doi: 10.1182/blood-2010-04-281360 PubMed DOI

Robb A, Wessling-Resnick M. Regulation of transferrin receptor 2 protein levels by transferrin. Blood. 2004;104: 4294–9. doi: 10.1182/blood-2004-06-2481 PubMed DOI

Pagani A, Vieillevoye M, Nai A, Rausa M, Ladli M, Lacombe C, et al. Regulation of cell surface transferrin receptor-2 by iron-dependent cleavage and release of a soluble form. Haematologica. 2015;100: 458–65. doi: 10.3324/haematol.2014.118521 PubMed DOI PMC

Gao J, Chen J, De Domenico I, Koeller DM, Harding CO, Fleming RE, et al. Hepatocyte-targeted HFE and TFR2 control hepcidin expression in mice. Blood. 2010;115: 3374–81. doi: 10.1182/blood-2009-09-245209 PubMed DOI PMC

Stirnberg M, Maurer E, Arenz K, Babler A, Jahnen-Dechent W, Gütschow M. Cell surface serine protease matriptase-2 suppresses fetuin-A/AHSG-mediated induction of hepcidin. Biol Chem. 2015;396: 81–93. doi: 10.1515/hsz-2014-0120 PubMed DOI

Jäckle F, Schmidt F, Wichert R, Arnold P, Prox J, Mangold M, et al. Metalloprotease meprin β is activated by transmembrane serine protease matriptase-2 at the cell surface thereby enhancing APP shedding. Biochem J. 2015;470: 91–103. doi: 10.1042/BJ20141417 PubMed DOI

Wahedi M, Wortham AW, Kleven MD, Zhao N, Jue S, Enns CA, et al. Matriptase-2 Suppresses Hepcidin Expression by Cleaving Multiple Components of the Hepcidin Induction Pathway. J Biol Chem. 2017. September 18. pii: jbc.M117.801795. doi: doi: 10.1074/jbc.M117.801795 PubMed DOI PMC

Rausa M, Ghitti M, Pagani A, Nai A, Campanella A, Musco G, et al. Identification of TMPRSS6 cleavage sites of hemojuvelin. J Cell Mol Med. 2015;19: 879–88. doi: 10.1111/jcmm.12462 PubMed DOI PMC

Nili M, Shinde U, Rotwein P. Soluble repulsive guidance molecule c/hemojuvelin is a broad spectrum bone morphogenetic protein (BMP) antagonist and inhibits both BMP2- and BMP6-mediated signaling and gene expression. J Biol Chem. 2010;285: 24783–92. doi: 10.1074/jbc.M110.130286 PubMed DOI PMC

Healey EG, Bishop B, Elegheert J, Bell CH, Padilla-Parra S, Siebold C. Repulsive guidance molecule is a structural bridge between neogenin and bone morphogenetic protein. Nat Struct Mol Biol. 2015;22: 458–65. doi: 10.1038/nsmb.3016 PubMed DOI PMC

Heart Ferroportin Protein Content Is Regulated by Heart Iron Concentration and Systemic Hepcidin Expression

Matriptase-2 and Hemojuvelin in Hepcidin Regulation: In Vivo Immunoblot Studies in Mask Mice

Effect of stimulated erythropoiesis on liver SMAD signaling pathway in iron-overloaded and iron-deficient mice