Matriptase-2 (TMPRSS6) is an important negative regulator of hepcidin expression; however, the effects of iron overload or accelerated erythropoiesis on liver TMPRSS6 protein content in vivo are largely unknown. We determined TMPRSS6 protein content in plasma membrane-enriched fractions of liver homogenates by immunoblotting, using a commercial antibody raised against the catalytic domain of TMPRSS6. Plasma membrane-enriched fractions were obtained by centrifugation at 3000 g and washing. TMPRSS6 was detected in the 3000 g fraction as a 120 kDa full-length protein in both mice and rats. Feeding of iron-deficient diet as well as erythropoietin treatment increased TMPRSS6 protein content in rats and mice by a posttranscriptional mechanism; the increase in TMPRSS6 protein by erythropoietin was also observed in Bmp6-mutant mice. Administration of high doses of iron to mice (200, 350 and 700 mg/kg) decreased TMPRSS6 protein content. Hemojuvelin was detected in the plasma membrane-enriched fractions of control animals as a full length protein of approximately 52 kDa; in iron deficient animals, the full length protein was partially cleaved at the N-terminus, resulting in an additional weak band of approximately 47 kDa. In livers from hemojuvelin-mutant mice, TMPRSS6 protein content was strongly decreased, suggesting that intact hemojuvelin is necessary for stable TMPRSS6 expression in the membrane. Overall, the results demonstrate posttranscriptional regulation of liver TMPRSS6 protein by iron status and erythropoietin administration, and provide support for the interaction of TMPRSS6 and hemojuvelin proteins in vivo.

Department of Pathology Kidney Group University Medical Center Utrecht Utrecht the Netherlands

Division of Medical Genetics Department of Medicine University of California San Diego La Jolla California United States of America

Institute of Biotechnology Laboratory of Tumour Resistance Academy of Sciences of the Czech Republic Prague Czech Republic

Institute of Pathophysiology Charles University Prague 1st Faculty of Medicine Prague Czech Republic

Zobrazit více v PubMed

Finberg KE. Iron-refractory iron deficiency anemia. Semin Hematol. 2009;46(4):378–86. 10.1053/j.seminhematol.2009.06.006 PubMed DOI

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. May 23;320(5879):1088–92. 10.1126/science.1157121 PubMed DOI PMC

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. May;40(5):569–71. 10.1038/ng.130 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. September 15;112(6):2539–45. 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(5):2089–91. 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. October;93(10):1473–9. 10.3324/haematol.13342 PubMed DOI

Ganz T. Systemic iron homeostasis. Physiol Rev. 2013;93(4):1721–41. 10.1152/physrev.00008.2013 PubMed DOI

Béliveau F, Désilets A, Leduc R. Probing the substrate specificities of matriptase, matriptase-2, hepsin and DESC1 with internally quenched fluorescent peptides. FEBS J. 2009;276(8):2213–26. 10.1111/j.1742-4658.2009.06950.x PubMed DOI

Silvestri L, Pagani A, Nai A, De Domenico I, Kaplan J, Camaschella C. The serine protease matripase-2 (TMPRSS6) inhibits hepcidin activation by cleaving membrane hemojuvelin. Cell Met. 2008;8(6):502–511. PubMed 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(4):1503–9. 10.1182/blood-2008-03-143354 PubMed DOI

Babitt JL, Huang FW, Wrighting DM, Xia Y, Sidis Y, Samad TA, et al. Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression. Nat Genet. 2006;38(5):531–9. PubMed

Truksa J, Gelbart T, Peng H, Beutler E, Beutler B, Lee P. Suppression of the hepcidin-encoding gene Hamp permits iron overload in mice lacking both hemojuvelin and matriptase-2/TMPRSS6. Br J Haematol. 2009;147(4):571–81. 10.1111/j.1365-2141.2009.07873.x PubMed DOI

Zhang AS, Anderson SA, Wang J, Yang F, DeMaster K, Ahmed R, et al. Suppression of hepatic hepcidin expression in response to acute iron deprivation is associated with an increase of matriptase-2 protein. Blood. 2011;117(5):1687–99. 10.1182/blood-2010-06-287292 PubMed DOI PMC

Lakhal S, Schödel J, Townsend AR, Pugh CW, Ratcliffe PJ, Mole DR. Regulation of type II transmembrane serine proteinase TMPRSS6 by hypoxia-inducible factors: new link between hypoxia signaling and iron homeostasis. J Biol Chem.2011;286(6):4090–7. 10.1074/jbc.M110.173096 PubMed DOI PMC

Maurer E, Gütschow M, Stirnberg M. Matriptase-2 (TMPRSS6) is directly up-regulated by hypoxia inducible factor-1: identification of a hypoxia-responsive element in the TMPRSS6 promoter region. Biol Chem. 2012;393(6):535–40. 10.1515/hsz-2011-0221 PubMed DOI

Meynard D, Vaja V, Sun CC, Corradini E, Chen S, López-Otín C, et al. Regulation of TMPRSS6 by BMP6 and iron in human cells and mice. Blood. 2011;118(3):747–56. 10.1182/blood-2011-04-348698 PubMed DOI PMC

Daba A, Gkouvatsos K, Sebastiani G, Pantopoulos K. Differences in activation of mouse hepcidin by dietary iron and parenterally administered iron dextran: compartmentalization is critical for iron sensing. J Mol Med (Berl). 2013;91(1):95–102. PubMed

Niederkofler V, Salie R, Arber S. Hemojuvelin is essential for dietary iron sensing, and its mutation leads to severe iron overload. J Clin Invest. 2005;115(8):2180–6. PubMed PMC

Dendooven A, van Oostrom O, van der Giezen DM, Leeuwis JW, Snijckers C, Joles JA, et al. Loss of endogenous bone morphogenetic protein-6 aggravates renal fibrosis. Am J Pathol. 2011;178(3):1069–79. 10.1016/j.ajpath.2010.12.005 PubMed DOI PMC

Wisniewski JR. Protocol to enrich and analyze plasma membrane proteins In: Peirce MJ, Wait R, editors. Membrane Proteomics: Methods and Protocols, vol. 528 Humana Press; 2009. p. 127–134. PubMed

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(4):405–11. PubMed

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

Krijt J, Fujikura Y, Ramsay AJ, Velasco G, Nečas E. Liver hemojuvelin protein levels in mice deficient in matriptase-2 (TMPRSS6). Blood Cells Mol Dis. 2011;47(2):133–7. PubMed

Maxson JE, Enns CA, Zhang AS. Processing of hemojuvelin requires retrograde trafficking to the Golgi in HepG2 cells. Blood. 2009;113(8):1786–93. 10.1182/blood-2008-08-174565 PubMed DOI PMC

Enns CA, Ahmed R, Zhang AS. Neogenin interacts with matriptase-2 to facilitate hemojuvelin cleavage. J Biol Chem. 2012;287(42):35104–17. 10.1074/jbc.M112.363937 PubMed DOI PMC

Ramsay AJ, Hooper JD, Folgueras AR, Velasco G, López-Otín C. Matriptase-2 (TMPRSS6): a proteolytic regulator of iron homeostasis. Haematologica. 2009;94(6):840–9. 10.3324/haematol.2008.001867 PubMed DOI PMC

Velasco G, Cal S, Quesada V, Sánchez LM, López-Otín C. Matriptase-2, a membrane-bound mosaic serine proteinase predominantly expressed in human liver and showing degrading activity against extracellular matrix proteins. J Biol Chem. 2002;277(40):37637–46. PubMed

Jiang J, Yang J, Feng P, Zuo B, Dong N, Wu Q, et al. N-glycosylation is required for matriptase-2 autoactivation and ectodomain shedding. J Biol Chem. 2014;289(28):19500–7. 10.1074/jbc.M114.555110 PubMed DOI PMC

Meynard D, Kautz L, Darnaud V, Canonne-Hergaux F, Coppin H, Roth MP. Lack of the bone morphogenetic protein BMP6 induces massive iron overload. Nat Genet. 2009;41(4):478–81. 10.1038/ng.320 PubMed DOI

Andriopoulos B Jr, Corradini E, Xia Y, Faasse SA, Chen S, Grgurevic L, et al. BMP6 is a key endogenous regulator of hepcidin expression and iron metabolism. Nat Genet. 2009;41(4):482–7. 10.1038/ng.335 PubMed DOI PMC

Ramos E, Kautz L, Rodriguez R, Hansen M, Gabayan V, Ginzburg Y, et al. Evidence for distinct pathways of hepcidin regulation by acute and chronic iron loading in mice. Hepatology. 2011;53(4):1333–41. 10.1002/hep.24178 PubMed DOI PMC

Corradini E, Meynard D, Wu Q, Chen S, Ventura P, Pietrangelo A, et al. Serum and liver iron differently regulate the bone morphogenetic protein 6 (BMP6)-SMAD signaling pathway in mice. Hepatology. 2011;54(1):273–84. 10.1002/hep.24359 PubMed DOI PMC

McDonald CJ, Wallace DF, Ostini L, Subramaniam VN. Parenteral vs. oral iron: influence on hepcidin signaling pathways through analysis of Hfe/Tfr2-null mice. Am J Physiol Gastrointest Liver Physiol. 2014;306(2):G132–9. 10.1152/ajpgi.00256.2013 PubMed DOI

Krijt J, Frýdlová J, Kukačková L, Fujikura Y, Přikryl P, Vokurka M, et al. Effect of iron overload and iron deficiency on liver hemojuvelin protein. PLoS One. 2012;7(5):e37391 10.1371/journal.pone.0037391 PubMed DOI PMC

Lee DH, Zhou LJ, Zhou Z, Xie JX, Jung JU, Liu Y, et al. Neogenin inhibits HFE2 secretion and regulates BMP-induced hepcidin expression and iron homeostasis. Blood. 2010;115(15):3136–45. 10.1182/blood-2009-11-251199 PubMed DOI PMC

Zhao N, Nizzi CP, Anderson SA, Wang J, Ueno A, Tsukamoto H, et al. Low intracellular iron increases the stability of matriptase-2. J Biol Chem. 2015;290(7):4432–46. 10.1074/jbc.M114.611913 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(32):24783–92. 10.1074/jbc.M110.130286 PubMed DOI PMC

Nicolas G, Viatte L, Bennoun M, Beaumont C, Kahn A, Vaulont S. Hepcidin, a new iron regulatory peptide. Blood Cells Mol Dis. 2002;29(3):327–35. PubMed

Krijt J, Jonášová A, Neuwirtová R, Nečas E. Effect of erythropoietin on hepcidin expression in hemojuvelin-mutant mice. Blood Cells Mol Dis. 2010;44(4):257–61. 10.1016/j.bcmd.2010.02.012 PubMed DOI

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(7):678–84. 10.1038/ng.2996 PubMed DOI PMC

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. February;152(4):498–500. 10.1111/j.1365-2141.2010.08473.x PubMed DOI

Lee P. EPO-mediated reduction in Hamp expression in vivo corrects iron deficiency anemia in TMPRSS6 deficiency [erratum]. Br J Haematol. 2012;156(3):415–6. PubMed 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(3):387–94. 10.1007/s00277-012-1618-8 PubMed DOI

Finch C. Regulators of iron balance in humans. Blood. 1994;84(6):1697–702. PubMed

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(21):5021–9. 10.1182/blood-2012-01-401885 PubMed DOI PMC

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(3):441–4. 10.1182/blood-2014-01-551150 PubMed DOI

Keel SB, Doty R, Liu L, Nemeth E, Cherian S, Ganz T, et al. Evidence that the expression of transferrin receptor 1 on erythroid marrow cells mediates hepcidin suppression in the liver. Exp Hematol. 2015;43(6):469–78. 10.1016/j.exphem.2015.03.001 PubMed DOI PMC

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

Effect of erythropoietin administration on proteins participating in iron homeostasis in Tmprss6-mutated mask mice