Železo a regulace jeho metabolismu při zánětu a poruchách erytropoézy.

• Název práce v češtině: Železo a regulace jeho metabolismu při zánětu a poruchách erytropoézy.
• Název v anglickém jazyce: Iron and regulation of its metabolism in inflammation and disorders of erythropoiesis.
• Klíčová slova: železo, hepcidin, erytropoetin, matriptáza-2, erytroferron, zánět
• Klíčová slova anglicky: iron, hepcidin, erythropoietin, matriptase-2, erythroferrone, inflammation
• Akademický rok vypsání: 2013/2014
• Typ práce: disertační práce
• Jazyk práce: čeština
• Ústav: Ústav patologické fyziologie 1. LF UK (11-00180)
• Vedoucí / školitel: prof. MUDr. Martin Vokurka, CSc.
• Řešitel: skrytý - zadáno a potvrzeno stud. odd.
• Datum přihlášení: 11.08.2014
• Datum zadání: 11.08.2014
• Datum potvrzení stud. oddělením: 11.08.2014
• Datum a čas obhajoby: 13.06.2019 10:00
• Místo konání obhajoby: Fyziologický ústav 1 .LF UK, Albertov 5, 128 00, Praha 2
• Datum odevzdání elektronické podoby: 27.03.2019
• Datum proběhlé obhajoby: 13.06.2019
• Předmět: Obhajoba dizertační práce (B90002)
• Oponenti: prof. MUDr. Kateřina Kaňková, Ph.D.
• Konzultanti: Ing. Jan Krijt, Ph.D.

Seznam odborné literatury

AREZES J, FOY N, MCHUGH K, SAWANT A, QUINKERT D, TERRAUBE V, BRINTH A, TAM M, LAVALLIE ER, TAYLOR S, ARMITAGE AE, PASRICHA SR, CUNNINGHAM O, LAMBERT M, DRAPER SJ, JASUJA R, DRAKESMITH H: Erythroferrone inhibits the induction of hepcidin by BMP6. Blood.132: 1473-1477, 2018.
ARTUSO I, PETTINATO M, NAI A, PAGANI A, SARDO U, BILLORE B, LIDONNICI MR, BENNETT C, MANDELLI G, PASRICHA SR, FERRARI G, CAMASCHELLA C, KAUTZ L, SILVESTRI L: Transient decrease of serum iron after acute erythropoietin treatment contributes to hepcidin inhibition by ERFE in mice. Haematologica.104: e87-e90, 2019.
BABITT JL, HUANG FW, WRIGHTING DM, XIA Y, SIDIS Y, SAMAD TA, CAMPAGNA JA, CHUNG RT, SCHNEYER AL, WOOLF CJ, ANDREWS NC, LIN HY: Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression. Nat Genet.38: 531-539, 2006.
BRIDLE KR, FRAZER DM, WILKINS SJ, DIXON JL, PURDIE DM, CRAWFORD DH, SUBRAMANIAM VN, POWELL LW, ANDERSON GJ, RAMM GA: Disrupted hepcidin regulation in HFE-associated haemochromatosis and the liver as a regulator of body iron homoeostasis. Lancet.361: 669-673, 2003.
BRISSOT P, CAVEY T, ROPERT M, GUGGENBUHL P, LOREAL O: Genetic hemochromatosis: Pathophysiology, diagnostic and therapeutic management. Presse Med.46: e288-e295, 2017.
COFFEY R, SARDO U, KAUTZ L, GABAYAN V, NEMETH E, GANZ T: Erythroferrone is not required for the glucoregulatory and hematologic effects of chronic erythropoietin treatment in mice. Physiol Rep.6: e13890, 2018.
DU X, SHE E, GELBART T, TRUKSA J, LEE P, XIA Y, KHOVANANTH K, MUDD S, MANN N, MORESCO EM, BEUTLER E, BEUTLER B: The serine protease TMPRSS6 is required to sense iron deficiency. Science.320: 1088-1092, 2008.
FEDER JN, GNIRKE A, THOMAS W, TSUCHIHASHI Z, RUDDY DA, BASAVA A, DORMISHIAN F, DOMINGO R, JR., ELLIS MC, FULLAN A, HINTON LM, JONES NL, KIMMEL BE, KRONMAL GS, LAUER P, LEE VK, LOEB DB, MAPA FA, MCCLELLAND E, MEYER NC, MINTIER GA, MOELLER N, MOORE T, MORIKANG E, PRASS CE, QUINTANA L, STARNES SM, SCHATZMAN RC, BRUNKE KJ, DRAYNA DT, RISCH NJ, BACON BR, WOLFF RK: A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nat Genet.13: 399-408, 1996.
FINBERG KE, HEENEY MM, CAMPAGNA DR, AYDINOK Y, PEARSON HA, HARTMAN KR, MAYO MM, SAMUEL SM, STROUSE JJ, MARKIANOS K, ANDREWS NC, FLEMING MD: Mutations in TMPRSS6 cause iron-refractory iron deficiency anemia (IRIDA). Nat Genet.40: 569-571, 2008.
FINCH C: Regulators of iron balance in humans. Blood.84: 1697-1702, 1994.
FOLGUERAS AR, DE LARA FM, PENDAS AM, GARABAYA C, RODRIGUEZ F, ASTUDILLO A, BERNAL T, CABANILLAS R, LOPEZ-OTIN C, VELASCO G: Membrane-bound serine protease matriptase-2 (Tmprss6) is an essential regulator of iron homeostasis. Blood.112: 2539-2545, 2008.
HEENEY MM, FINBERG KE: Iron-refractory iron deficiency anemia (IRIDA). Hematol Oncol Clin North Am.28: 637-652, v, 2014.
KAUTZ L, JUNG G, VALORE EV, RIVELLA S, NEMETH E, GANZ T: Identification of erythroferrone as an erythroid regulator of iron metabolism. Nat Genet.46: 678-684, 2014b.
KAUTZ L, MEYNARD D, MONNIER A, DARNAUD V, BOUVET R, WANG RH, DENG C, VAULONT S, MOSSER J, COPPIN H, ROTH MP: Iron regulates phosphorylation of Smad1/5/8 and gene expression of Bmp6, Smad7, Id1, and Atoh8 in the mouse liver. Blood.112: 1503-1509, 2008.
KIM A, NEMETH E: New insights into iron regulation and erythropoiesis. Curr Opin Hematol.22: 199-205, 2015.
KRIJT J, JONASOVA A, NEUWIRTOVA R, NECAS E: Effect of erythropoietin on hepcidin expression in hemojuvelin-mutant mice. Blood Cells Mol Dis.44: 257-261, 2010b.
LENOX LE, SHI L, HEGDE S, PAULSON RF: Extramedullary erythropoiesis in the adult liver requires BMP-4/Smad5-dependent signaling. Exp Hematol.37: 549-558, 2009.
MIYAKE T, KUNG CK, GOLDWASSER E: Purification of human erythropoietin. J Biol Chem.252: 5558-5564, 1977.
MUCKENTHALER MU, RIVELLA S, HENTZE MW, GALY B: A Red Carpet for Iron Metabolism. Cell.168: 344-361, 2017.
NEMETH E, GANZ T: Anemia of inflammation. Hematol Oncol Clin North Am.28: 671-681, vi, 2014.
NEMETH E, RIVERA S, GABAYAN V, KELLER C, TAUDORF S, PEDERSEN BK, GANZ T: IL-6 mediates hypoferremia of inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. J Clin Invest.113: 1271-1276, 2004a.
NEMETH E, VALORE EV, TERRITO M, SCHILLER G, LICHTENSTEIN A, GANZ T: Hepcidin, a putative mediator of anemia of inflammation, is a type II acute-phase protein. Blood.101: 2461-2463, 2003.
NICOLAS G, BENNOUN M, DEVAUX I, BEAUMONT C, GRANDCHAMP B, KAHN A, VAULONT S: 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.98: 8780-8785, 2001.
NICOLAS G, BENNOUN M, PORTEU A, MATIVET S, BEAUMONT C, GRANDCHAMP B, SIRITO M, SAWADOGO M, KAHN A, VAULONT S: Severe iron deficiency anemia in transgenic mice expressing liver hepcidin. Proc Natl Acad Sci U S A.99: 4596-4601, 2002a.
NICOLAS G, CHAUVET C, VIATTE L, DANAN JL, BIGARD X, DEVAUX I, BEAUMONT C, KAHN A, VAULONT S: The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. J Clin Invest.110: 1037-1044, 2002b.
NICOLAS G, VIATTE L, BENNOUN M, BEAUMONT C, KAHN A, VAULONT S: Hepcidin, a new iron regulatory peptide. Blood Cells Mol Dis.29: 327-335, 2002c.
NICOLAS G, DESCHEMIN JC, RAMSAY AJ, MAYEUX P, GRANDCHAMP B, BEAUMONT C, VELASCO G, VAULONT S: Is EPO therapy able to correct iron deficiency anaemia caused by matriptase-2 deficiency? Br J Haematol.152: 498-500, 2011.
NIEDERAU C, FISCHER R, PURSCHEL A, STREMMEL W, HAUSSINGER D, STROHMEYER G: Long-term survival in patients with hereditary hemochromatosis. Gastroenterology.110: 1107-1119, 1996.
PAK M, LOPEZ MA, GABAYAN V, GANZ T, RIVERA S: Suppression of hepcidin during anemia requires erythropoietic activity. Blood.108: 3730-3735, 2006.
PIGEON C, ILYIN G, COURSELAUD B, LEROYER P, TURLIN B, BRISSOT P, LOREAL O: A new mouse liver-specific gene, encoding a protein homologous to human antimicrobial peptide hepcidin, is overexpressed during iron overload. J Biol Chem.276: 7811-7819, 2001.
RAVASI G, PELUCCHI S, BUOLI COMANI G, GRENI F, MARIANI R, PELLONI I, BOMBELLI S, PEREGO R, BARISANI D, PIPERNO A: Hepcidin regulation in a mouse model of acute hypoxia. Eur J Haematol.100: 636-643, 2018.
RISHI G, SUBRAMANIAM VN: The relationship between systemic iron homeostasis and erythropoiesis. Biosci Rep.37, 2017.
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.287: 11968-11980, 2012.
VOKURKA M, KRIJT J, SULC K, NECAS E: Hepcidin mRNA levels in mouse liver respond to inhibition of erythropoiesis. Physiol Res.55: 667-674, 2006.
VOKURKA M, LACINOVA Z, KREMEN J, KOPECKY P, BLAHA J, PELINKOVA K, HALUZIK M, NECAS E: Hepcidin expression in adipose tissue increases during cardiac surgery. Physiol Res.59: 393-400, 2010.
WANG RH, LI C, XU X, ZHENG Y, XIAO C, ZERFAS P, COOPERMAN S, ECKHAUS M, ROUAULT T, MISHRA L, DENG CX: A role of SMAD4 in iron metabolism through the positive regulation of hepcidin expression. Cell Metab.2: 399-409, 2005.
WEISS G, GOODNOUGH LT: Anemia of chronic disease. N Engl J Med.352: 1011-1023, 2005.
WRIGHTING DM, ANDREWS NC: Interleukin-6 induces hepcidin expression through STAT3. Blood.108: 3204-3209, 2006.

Předběžná náplň práce

Cílem naší práce bylo zjistit, jak je MT-2 zapojena do erytroidní dráhy a zdali nemůže být místem, kde se protínají jednotlivé regulační dráhy. Stanovovali jsme expresi genů na úrovni mRNA pomocí real-time PCR a na úrovni proteinů pomocí imunoblotů. K pokusům jsme nejprve použili samce myši a samice potkanů, kterým byl podáván erytropoetin (EPO), železo či byli na nízkoželezné dietě. Prokázali jsme, že železo ani EPO neovlivňují expresi MT-2 na úrovni mRNA, ale že je množství MT-2 ovlivněno na úrovni proteinu. EPO a nedostatek železa navozený nízkoželeznou dietou ji zvyšují, podání železa ji naopak snižuje. Změny byly výraznější u potkanů. V dalších experimentech jsme proto u potkanů podali kombinaci podávání železa a EPO a sledovali jejich vliv na MT-2 aERFE. Potvrdili jsme předešlé pozorování na myších, že při předchozím podání vysokých dávek železa nedochází po podání EPO k snížení exprese hepcidinu, a to přesto, že exprese ERFE ve slezině zůstává zvýšena. Protože podání železa zabránilo vzestupu množství proteinu MT-2 po EPO, domníváme se, že MT-2 přispívá k omezení poklesu hepcidinu.

Předběžná náplň práce v anglickém jazyce

The aim of our work was to investigate how MT-2 is involved in the erythroid regulatory pathway, and whether it can represent the molecule where various regulatory pathways interact. We determined gene expression as the amount of mRNA by real-time PCR and as protein level by immunoblots. Firstly, we used male mice and female rats which were given erythropoietin (EPO), iron or were kept on low iron diet. We proved that neither iron nor EPO influenced MT-2 expression on the mRNA level but that MT-2 reacted to both on protein level. EPO and iron deficiency induced by low iron diet increased the amount of MT-2 protein while iron administration decreased it. The changes were more pronounced in rats. In next experiments we therefore administered the combination of iron and EPO and studied their influence on MT-2 and ERFE in rats. We confirmed previous data in mice that the previous administration of high iron doses blocks the EPO mediated downregulation of hepcidin expression despite the increased ERFE expression in the spleen. As iron administration prevented the increase of MT-2 protein amount observed after EPO, we suppose that MT-2 plays role in the limitation of hepcidin downregulation.