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Hepcidin suppression in β-thalassemia is associated with the down-regulation of atonal homolog 8

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Abstract

Atonal homolog 8 (ATOH8) is defined as a positive regulator of hepcidin transcription, which links erythropoietic activity with iron-sensing molecules. In the present study, we investigated the association between hepcidin and ATOH8 expression in β-thalassemia. We found that inhibition of hepcidin expression in β-thalassemia is correlated with reduced ATOH8 expression. Hepatic hepcidin 1 (Hamp1) and Atoh8 mRNA expression were down-regulated in β-thalassemic mice. Hepcidin (HAMP) and ATOH8 mRNA expression were consistently suppressed in Huh7 cells cultured in medium supplemented with β-thalassemia patient serum. The Huh7 cells, which were transfected with ATOH8-FLAG expression plasmid and cultured in the supplemented medium, exhibited increased levels of ATOH8 mRNA, ATOH8-FLAG protein, pSMAD1,5,8, and HAMP mRNA. Interestingly, over-expression of ATOH8 reversed the effects of hepcidin suppression induced by the β-thalassemia patient sera. In conclusion, hepcidin suppression in β-thalassemia is associated with the down-regulation of ATOH8 in response to anemia. We, therefore, suggest that ATOH8 is an important transcriptional regulator of hepcidin in β-thalassemia.

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References

  1. Park CH, Valore EV, Waring AJ, Ganz T. Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J Biol Chem. 2001;276:7806–10.

    Article  CAS  PubMed  Google Scholar 

  2. Atanasiu V, Manolescu B, Stoian I. Hepcidin–central regulator of iron metabolism. Eur J Haematol. 2007;78:1–10.

    Article  CAS  PubMed  Google Scholar 

  3. Leong WI, Lonnerdal B. Hepcidin, the recently identified peptide that appears to regulate iron absorption. J Nutr. 2004;134:1–4.

    CAS  PubMed  Google Scholar 

  4. Rossi E. Hepcidin–the iron regulatory hormone. Clin Biochem Rev. 2005;26:47–9.

    PubMed  PubMed Central  Google Scholar 

  5. Ganz T, Nemeth E. Iron imports. IV. Hepcidin and regulation of body iron metabolism. Am J Physiol Gastrointest Liver Physiol. 2006;290:G199–203.

    Article  CAS  PubMed  Google Scholar 

  6. 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.

    Article  CAS  PubMed  Google Scholar 

  7. McKie AT, Marciani P, Rolfs A, Brennan K, Wehr K, Barrow D, et al. A novel duodenal iron-regulated transporter, IREG1, implicated in the basolateral transfer of iron to the circulation. Mol Cell. 2000;5:299–309.

    Article  CAS  PubMed  Google Scholar 

  8. Abboud S, Haile DJ. A novel mammalian iron-regulated protein involved in intracellular iron metabolism. J Biol Chem. 2000;275:19906–12.

    Article  CAS  PubMed  Google Scholar 

  9. Donovan A, Brownlie A, Zhou Y, Shepard J, Pratt SJ, Moynihan J, et al. Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter. Nature. 2000;403:776–81.

    Article  CAS  PubMed  Google Scholar 

  10. Knutson MD, Oukka M, Koss LM, Aydemir F, Wessling-Resnick M. Iron release from macrophages after erythrophagocytosis is up-regulated by ferroportin 1 overexpression and down-regulated by hepcidin. Proc Natl Acad Sci USA. 2005;102:1324–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 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;41:478–81.

    Article  CAS  PubMed  Google Scholar 

  12. 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;53:1333–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Wrighting DM, Andrews NC. Interleukin-6 induces hepcidin expression through STAT3. Blood. 2006;108:3204–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Fung E, Nemeth E. Manipulation of the hepcidin pathway for therapeutic purposes. Haematologica. 2013;98:1667–76.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Hershko C. Iron loading and its clinical implications. Am J Hematol. 2007;82:1147–8.

    Article  CAS  PubMed  Google Scholar 

  16. Tanno T, Bhanu NV, Oneal PA, Goh SH, Staker P, Lee YT, et al. High levels of GDF15 in thalassemia suppress expression of the iron regulatory protein hepcidin. Nat Med. 2007;13:1096–101.

    Article  CAS  PubMed  Google Scholar 

  17. Tanno T, Porayette P, Sripichai O, Noh SJ, Byrnes C, Bhupatiraju A, et al. Identification of TWSG1 as a second novel erythroid regulator of hepcidin expression in murine and human cells. Blood. 2009;114:181–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Origa R, Galanello R, Ganz T, Giagu N, Maccioni L, Faa G, et al. Liver iron concentrations and urinary hepcidin in beta-thalassemia. Haematologica. 2007;92:583–8.

    Article  CAS  PubMed  Google Scholar 

  20. Jones E, Pasricha SR, Allen A, Evans P, Fisher CA, Wray K, et al. Hepcidin is suppressed by erythropoiesis in hemoglobin E beta-thalassemia and beta-thalassemia trait. Blood. 2015;125:873–80.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Camberlein E, Zanninelli G, Detivaud L, Lizzi AR, Sorrentino F, Vacquer S, et al. Anemia in beta-thalassemia patients targets hepatic hepcidin transcript levels independently of iron metabolism genes controlling hepcidin expression. Haematologica. 2008;93:111–5.

    Article  CAS  PubMed  Google Scholar 

  22. Kearney SL, Nemeth E, Neufeld EJ, Thapa D, Ganz T, Weinstein DA, et al. Urinary hepcidin in congenital chronic anemias. Pediatr Blood Cancer. 2007;48:57–63.

    Article  PubMed  Google Scholar 

  23. Nemeth E, Ganz T. Hepcidin and iron-loading anemias. Haematologica. 2006;91:727–32.

    CAS  PubMed  Google Scholar 

  24. Patel N, Varghese J, Masaratana P, Latunde-Dada GO, Jacob M, Simpson RJ, et al. The transcription factor ATOH8 is regulated by erythropoietic activity and regulates HAMP transcription and cellular pSMAD1,5,8 levels. Br J Haematol. 2014;164:586–96.

    Article  CAS  PubMed  Google Scholar 

  25. Frazer DM, Wilkins SJ, Darshan D, Badrick AC, McLaren GD, Anderson GJ. Stimulated erythropoiesis with secondary iron loading leads to a decrease in hepcidin despite an increase in bone morphogenetic protein 6 expression. Br J Haematol. 2012;157:615–26.

    Article  CAS  PubMed  Google Scholar 

  26. Jamsai D, Zaibak F, Khongnium W, Vadolas J, Voullaire L, Fowler KJ, et al. A humanized mouse model for a common beta0-thalassemia mutation. Genomics. 2005;85:453–61.

    Article  CAS  PubMed  Google Scholar 

  27. Jamsai D, Zaibak F, Vadolas J, Voullaire L, Fowler KJ, Gazeas S, et al. A humanized BAC transgenic/knockout mouse model for HbE/beta-thalassemia. Genomics. 2006;88:309–15.

    Article  CAS  PubMed  Google Scholar 

  28. Ma Y, Podinovskaia M, Evans PJ, Emma G, Schaible UE, Porter J, et al. A novel method for non-transferrin-bound iron quantification by chelatable fluorescent beads based on flow cytometry. Biochem J. 2014;463:351–62.

    Article  CAS  PubMed  Google Scholar 

  29. Upanan S, Pangjit K, Uthaipibull C, Fucharoen S, McKie AT, Srichairatanakool S. Combined treatment of 3-hydroxypyridine-4-one derivatives and green tea extract to induce hepcidin expression in iron-overloaded β-thalassemic mice. Asian Pac J Trop Biomed. 2015;5:1010–7.

    Article  Google Scholar 

  30. Weizer-Stern O, Adamsky K, Amariglio N, Levin C, Koren A, Breuer W, et al. Downregulation of hepcidin and haemojuvelin expression in the hepatocyte cell-line HepG2 induced by thalassaemic sera. Br J Haematol. 2006;135:129–38.

    Article  CAS  PubMed  Google Scholar 

  31. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25:402–8.

    Article  CAS  PubMed  Google Scholar 

  32. Patel N, Masaratana P, Diaz-Castro J, Latunde-Dada GO, Qureshi A, Lockyer P, et al. BMPER protein is a negative regulator of hepcidin and is up-regulated in hypotransferrinemic mice. J Biol Chem. 2012;287:4099–106.

    Article  CAS  PubMed  Google Scholar 

  33. Casanovas G, Vujic Spasic M, Casu C, Rivella S, Strelau J, Unsicker K, et al. The murine growth differentiation factor 15 is not essential for systemic iron homeostasis in phlebotomized mice. Haematologica. 2013;98:444–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Parrow NL, Gardenghi S, Ramos P, Casu C, Grady RW, Anderson ER, et al. Decreased hepcidin expression in murine beta-thalassemia is associated with suppression of Bmp/Smad signaling. Blood. 2012;119:3187–9.

    Article  CAS  PubMed  Google Scholar 

  35. Weizer-Stern O, Adamsky K, Amariglio N, Rachmilewitz E, Breda L, Rivella S, et al. mRNA expression of iron regulatory genes in beta-thalassemia intermedia and beta-thalassemia major mouse models. Am J Hematol. 2006;81:479–83.

    Article  CAS  PubMed  Google Scholar 

  36. Gardenghi S, Marongiu MF, Ramos P, Guy E, Breda L, Chadburn A, et al. Ineffective erythropoiesis in beta-thalassemia is characterized by increased iron absorption mediated by down-regulation of hepcidin and up-regulation of ferroportin. Blood. 2007;109:5027–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Adamsky K, Weizer O, Amariglio N, Breda L, Harmelin A, Rivella S, et al. Decreased hepcidin mRNA expression in thalassemic mice. Br J Haematol. 2004;124:123–4.

    Article  CAS  PubMed  Google Scholar 

  38. De Franceschi L, Daraio F, Filippini A, Carturan S, Muchitsch EM, Roetto A, et al. Liver expression of hepcidin and other iron genes in two mouse models of beta-thalassemia. Haematologica. 2006;91:1336–42.

    PubMed  Google Scholar 

  39. Breda L, Gardenghi S, Guy E, Rachmilewitz EA, Weizer-Stern O, Adamsky K, et al. Exploring the role of hepcidin, an antimicrobial and iron regulatory peptide, in increased iron absorption in beta-thalassemia. Ann N Y Acad Sci. 2005;1054:417–22.

    Article  CAS  PubMed  Google Scholar 

  40. Frazer DM, Inglis HR, Wilkins SJ, Millard KN, Steele TM, McLaren GD, et al. Delayed hepcidin response explains the lag period in iron absorption following a stimulus to increase erythropoiesis. Gut. 2004;53:1509–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Latunde-Dada GO, McKie AT, Simpson RJ. Animal models with enhanced erythropoiesis and iron absorption. Biochim Biophys Acta. 2006;1762:414–23.

    Article  CAS  PubMed  Google Scholar 

  42. Masaratana P, Latunde-Dada GO, Patel N, Simpson RJ, Vaulont S, McKie AT. Iron metabolism in hepcidin1 knockout mice in response to phenylhydrazine-induced hemolysis. Blood Cells Mol Dis. 2012;49:85–91.

    Article  CAS  PubMed  Google Scholar 

  43. Nicolas G, Chauvet C, Viatte L, Danan JL, Bigard X, Devaux I, et al. The gene encoding the iron regulatory peptide hepcidin is regulated by anemia, hypoxia, and inflammation. J Clin Invest. 2002;110:1037–44.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Pak M, Lopez MA, Gabayan V, Ganz T, Rivera S. Suppression of hepcidin during anemia requires erythropoietic activity. Blood. 2006;108:3730–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Kemna EH, Kartikasari AE, van Tits LJ, Pickkers P, Tjalsma H, Swinkels DW. Regulation of hepcidin: insights from biochemical analyses on human serum samples. Blood Cells Mol Dis. 2008;40:339–46.

    Article  CAS  PubMed  Google Scholar 

  46. Kaddah NA, El Gindi HD, Mostafa NO, Abd el aziz NMS, Kamhawy AHA. Role of hepcidin in the pathogenesis of iron overload in children with B-thalassemia. Inter J Aced Res. 2001;3:62–9.

    Google Scholar 

  47. Darshan D, Frazer DM, Anderson GJ. Molecular basis of iron-loading disorders. Expert Rev Mol Med. 2010;12:e36.

    Article  PubMed  Google Scholar 

  48. 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, Smad 7, Id1, and Atoh8 in the mouse liver. Blood. 2008;112:1503–9.

    Article  CAS  PubMed  Google Scholar 

  49. Bartnikas TB, Andrews NC, Fleming MD. Transferrin is a major determinant of hepcidin expression in hypotransferrinemic mice. Blood. 2011;117:630–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This research study received the financial support by the Royal Golden Jubilee PhD Program, Thailand Research Fund (Grant No. PHD/0345/2552); Faculty of Medicine Research Fund, Chiang Mai University, Thailand; and the Chair Professor Grant of National Science and Technology Development Agency through Prof. Suthat Fucharoen, MD. We thank the Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Thailand for supplying β-thalassemic mice. The authors acknowledge Dr. Neeta Patel (Division of Diabetes and Nutritional Sciences, King’s College London, UK) for providing us with the ATOH8-FLAG expression plasmid. We thank the Division of Diabetes and Nutritional Sciences, King’s College London, UK; the FWB Genomics Centre, King’s College London, UK; Thailand Excellence for Tissue Engineering and Stem Cells, Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Thailand; and the Medical Science Research Equipment Centre, Faculty of Medicine, Chiang Mai University, Thailand for supplying the research equipment. The histochemical examination of tissue iron was diagnosed and photographed by expert pathologists, Parichart Wongsena, MD and Mr. Taksakorn Wongseeda, College of Medicine and Public Health, Ubon Ratchathani University, Thailand.

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Authors and Affiliations

  1. Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai, 50200, Thailand

    Supranee Upanan, Peraphan Pothacharoen, Prachya Kongtawelert & Somdet Srichairatanakool

  2. Division of Diabetes and Nutritional Sciences, School of Medicine, King’s College London, London, SE1 9NH, UK

    Andrew T. McKie & Gladys O. Latunde-Dada

  3. National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Thailand Science Park, Pathum Thani, 12120, Thailand

    Sittiruk Roytrakul & Chairat Uthaipibull

  4. Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom, 73170, Thailand

    Suthat Fucharoen

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  1. Supranee Upanan
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12185_2017_2231_MOESM1_ESM.jpg

Fig. S1 Dose–response expression of ATOH8 and hepcidin in ATOH8-transfected Huh7 cells grown in β-thalassemia patient serum. Huh7 cells were transfected with ATOH8-FLAG expression plasmid (0, 0.5, 1, and 2 µg) and grown in DMEM+female HbE/T for 24 h. The cells were subjected to qPCR analysis for ATOH8 (a) and HAMP (b) mRNA expression. Relative mRNA expression was acquired by normalizing to RPL19. Values (mean ± SD) obtained from triplicate samples are expressed as fold changes compared with the group without ATOH8-FLAG expression plasmid. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. * p < 0.05. The whole cell lysates of biological triplicate samples were also subjected to western blot analysis to detect ATOH8-FLAG protein levels (c). HbE/T: HbE/β-thalassemia. (JPEG 94 kb)

12185_2017_2231_MOESM2_ESM.jpg

Fig. S2 Dose–response expression of pSMAD1,5,8 in ATOH8-transfected Huh7 cells grown in β-thalassemia patient serum. Huh7 cells were transfected with ATOH8-FLAG expression plasmid (0, 0.5, 1, and 2 µg) and grown in the DMEM+female HbE/T for 24 h. The whole cell lysates of biological triplicate samples were subjected to western blot analysis. Densitometry is displayed below the blot. pSMAD1,5,8 levels were normalized to total SMAD1,5,8 and β-actin and are expressed as fold changes (mean ± SD) compared with the group without ATOH8-FLAG expression plasmid. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. * p < 0.05. HbE/T: HbE/β-thalassemia. (JPEG 121 kb)

12185_2017_2231_MOESM3_ESM.jpg

Fig. S3 Dose–response expression of ATOH8 and hepcidin in ATOH8-transfected HEK293 cells grown in β-thalassemia patient serum. HEK293 cells were transfected with ATOH8-FLAG expression plasmid (0, 0.5, 1, and 2 µg) in DMEM for 6 h in the starvation step of cell culture and grown in the DMEM+female HbE/T for 24 h. The cells were subjected to qPCR analysis for ATOH8 (a) and HAMP (b) mRNA expression. Relative mRNA expression was acquired by normalizing to RPL19. Values (mean ± SD) obtained from triplicate samples are expressed as fold changes compared with the group without ATOH8-FLAG expression plasmid. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. * p < 0.05. The whole cell lysates of biological triplicate samples were also subjected to western blot analysis to detect ATOH8-FLAG protein levels (c). HbE/T: HbE/β-thalassemia. (JPEG 92 kb)

12185_2017_2231_MOESM4_ESM.jpg

Fig. S4 Dose–response expression of pSMAD1,5,8 in ATOH8-transfected HEK293 cells grown in β-thalassemia patient serum. HEK293 cells were transfected with ATOH8-FLAG expression plasmid (0, 0.5, 1, and 2 µg) in DMEM for 6 h in the starvation step of cell culture and grown in the DMEM+female HbE/T for 24 h. The whole cell lysates of biological triplicate samples were subjected to western blot analysis. Densitometry is displayed below the blot. pSMAD1,5,8 levels were normalized to total SMAD1,5,8 and β-actin and are expressed as fold changes (mean ± SD) compared with the group without ATOH8-FLAG expression plasmid. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test. * p < 0.05. HbE/T: HbE/β-thalassemia. (JPEG 126 kb)

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Upanan, S., McKie, A.T., Latunde-Dada, G.O. et al. Hepcidin suppression in β-thalassemia is associated with the down-regulation of atonal homolog 8. Int J Hematol 106, 196–205 (2017). https://doi.org/10.1007/s12185-017-2231-3

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