Molecular Neurobiology

, Volume 56, Issue 4, pp 2362–2374 | Cite as

Hepcidin Mediates Transcriptional Changes in Ferroportin mRNA in Differentiated Neuronal-Like PC12 Cells Subjected to Iron Challenge

  • Steinunn Sara Helgudottir
  • Jacek Lichota
  • Annette Burkhart
  • Torben MoosEmail author


Ferroportin is the only known iron exporter, and its regulation seems to be controlled at both transcriptional, post-transcriptional, and post-translational levels. The objective of the current work was to investigate how cellular iron status affects the expression of the ferroportin gene Fpn under the influence of hepcidin, known to post-translational lower the available ferroportin protein. Nerve growth factor-beta (NGF-β)-differentiated PC12 cells, used as a model of neuronal cells, were evaluated in terms of their viability and expression of ferroportin after inducing cellular iron overload with ferric ammonium citrate (FAC) or hepcidin, iron deficiency with deferoxamine (DFO), or hepcidin in combination with FAC or DFO. Ferritin mRNA was significantly upregulated following treatment with 20 mM FAC. The viability of the differentiated PC12 cells was significantly reduced after treatment with 30 mM FAC or 1.0 μM hepcidin, but when combining FAC and hepcidin treatment, the cells remained unaffected. The expression of Fpn was concurrently upregulated after treatment with FAC in combination with hepcidin. Fifty millimolar DFO also increased Fpn. Together, these data point towards a transcriptional induction of Fpn in response to changes in cellular iron levels. Epigenetic regulation of Fpn may also occur as changes in genes associated with epigenetic regulation of Fpn were demonstrated.


Ferroportin Iron Neurodegeneration Hepcidin Epigenetic 



Antioxidant response element


Bovine serum albumin




4′,6-Diamidino-2-phenylindole dihydrochloride




Divalent metal transporter 1


Days in vitro


Ferric ammonium iron (III) citrate


Fetal calf serum


Ferrous iron


Ferric iron


Ferroportin gene expression


Ferritin light chain


Ferritin heavy chain


Histone deacetylase 1


Iron-responsive element


Iron regulatory protein


Nerve growth factor beta1


Phosphate-buffered saline




PHD finger protein 8


Reactive nitrogen species


Reactive oxygen species


Standard error of the mean


Ten-eleven translocation methylcytosine dioxygenase 1


Class IV β-tubulin


Untranslated region



We thank Poul Henning Jensen for providing PC12 cells and Merete Fredsgaard and Hanne Krone Nielsen, Aalborg University, Denmark, for the excellent technical assistance. We thank Assistant Professor Maj Schneider Thomsen, Aalborg University, Denmark, for the illustrative work.

Funding Information

The present work has been supported by The Danish Multiple Sclerosis Society, “Fonden til Lægevidenskabens Fremme,” Augustinus fonden, and the “Åse og Ejner Danielsens Fond.”

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interests.


  1. 1.
    Belaidi AA, Bush AI (2016) Iron neurochemistry in Alzheimer’s disease and Parkinson’s disease: targets for therapeutics. J Neurochem 139:179–197. CrossRefPubMedGoogle Scholar
  2. 2.
    Moos T, Rosengren Nielsen T, Skjørringe T, Morgan EH (2007) Iron trafficking inside the brain. J Neurochem 103:1730–1740. CrossRefPubMedGoogle Scholar
  3. 3.
    Andersen HH, Johnsen KB, Moos T (2014) Iron deposits in the chronically inflamed central nervous system and contributes to neurodegeneration. Cell Mol Life Sci 71:1607–1622. CrossRefPubMedGoogle Scholar
  4. 4.
    Crichton RR, Dexter DT, Ward RJ (2011) Brain iron metabolism and its perturbation in neurological diseases. J Neural Transm 118:301–314. CrossRefPubMedGoogle Scholar
  5. 5.
    Ward RJ, Zucca FA, Duyn JH et al (2014) The role of iron in brain ageing and neurodegenerative disorders. Lancet Neurol 13:1045–1060CrossRefGoogle Scholar
  6. 6.
    Mills E, Dong X-P, Wang F, Xu H (2010) Mechanisms of brain iron transport: insight into neurodegeneration and CNS disorders. Future Med Chem 2:51–64. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Ward DM, Kaplan J (2012) Ferroportin-mediated iron transport: expression and regulation. Biochim Biophys Acta - Mol Cell Res 1823:1426–1433. CrossRefGoogle Scholar
  8. 8.
    Le Gac G, Ka C, Joubrel R et al (2013) Structure-function analysis of the human ferroportin iron exporter (SLC40A1): effect of hemochromatosis type 4 disease mutations and identification of critical residues. Hum Mutat 34:1371–1380. CrossRefPubMedGoogle Scholar
  9. 9.
    Bonaccorsi di Patti MC, Polticelli F, Cece G et al (2014) A structural model of human ferroportin and of its iron binding site. FEBS J 281:2851–2860. CrossRefPubMedGoogle Scholar
  10. 10.
    Rochette L, Gudjoncik A, Guenancia C et al (2015) The iron-regulatory hormone hepcidin: a possible therapeutic target? Pharmacol Ther 146:35–52. CrossRefPubMedGoogle Scholar
  11. 11.
    Guo W, Zhang S, Chen Y et al (2015) An important role of the hepcidin–ferroportin signaling in affecting tumor growth and metastasis. Acta Biochim Biophys Sin Shanghai 47:703–715. CrossRefPubMedGoogle Scholar
  12. 12.
    Drakesmith H, Nemeth E, Ganz T (2015) Ironing out ferroportin. Cell Metab 22:777–787. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Oates PS (2007) The role of hepcidin and ferroportin in iron absorption. Histol Histopathol 22:791–804PubMedGoogle Scholar
  14. 14.
    Wallace DF (2016) The regulation of iron absorption and homeostasis. Clin Biochem Rev 37:51–62Google Scholar
  15. 15.
    Marro S, Chiabrando D, Messana E et al (2010) Heme controls ferroportin1 (FPN1) transcription involving Bach1, Nrf2 and a MARE/ARE sequence motif at position -7007 of the FPN1 promoter. Haematologica 95:1261–1268. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Chung J, Haile DJ, Wessling-Resnick M (2004) Copper-induced ferroportin-1 expression in J774 macrophages is associated with increased iron efflux. Proc Natl Acad Sci U S A 101:2700–2705. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Silva B, Faustino P (2015) An overview of molecular basis of iron metabolism regulation and the associated pathologies. Biochim Biophys Acta 1852:1347–1359. CrossRefPubMedGoogle Scholar
  18. 18.
    Gulec S, Anderson GJ, Collins JF (2014) Mechanistic and regulatory aspects of intestinal iron absorption. AJP Gastrointest Liver Physiol 307:G397–G409. CrossRefGoogle Scholar
  19. 19.
    Burkhart A, Skjørringe T, Johnsen KB, Moos T (2015) Divalent metal transporter 1 (DMT1) in the brain: implications for a role in iron transport at the blood-brain barrier, and neuronal and glial pathology. Front Mol Neurosci 8:19. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Chen Y, Zhang S, Wang X et al (2015) Disordered signaling governing ferroportin transcription favors breast cancer growth. Cell Signal 27:168–176. CrossRefPubMedGoogle Scholar
  21. 21.
    Szyf M (2015) Prospects for the development of epigenetic drugs for CNS conditions. Nat Rev Drug Discov 14:461–474. CrossRefPubMedGoogle Scholar
  22. 22.
    Yao B, Christian KM, He C et al (2016) Epigenetic mechanisms in neurogenesis. Nat Rev Neurosci 17:537–549. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Arrowsmith CH, Bountra C, Fish PV et al (2012) Epigenetic protein families: a new frontier for drug discovery. Nat Rev Drug Discov 11:384–400. CrossRefPubMedGoogle Scholar
  24. 24.
    Qureshi IA, Mehler MF (2015) Epigenetics and therapeutic targets mediating neuroprotection. Brain Res 1628:265–272. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Karuppagounder SS, Kumar A, Shao DS et al (2015) Metabolism and epigenetics in the nervous system: creating cellular fitness and resistance to neuronal death in neurological conditions via modulation of oxygen-, iron-, and 2-oxoglutarate-dependent dioxygenases. Brain Res 1628:1–15. CrossRefGoogle Scholar
  26. 26.
    Guo JU, Su Y, Zhong C et al (2011) Hydroxylation of 5-methylcytosine by TET1 promotes active DNA demethylation in the adult brain. Cell 145:423–434. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Das KP, Freudenrich TM, Mundy WR (2004) Assessment of PC12 cell differentiation and neurite growth: a comparison of morphological and neurochemical measures. Neurotoxicol Teratol 26:397–406. CrossRefPubMedGoogle Scholar
  28. 28.
    Yang H, Cabral F (2007) Heightened sensitivity to paclitaxel in class IVa β-tubulin-transfected cells is lost as expression increases. J Biol Chem 282:27058–27066. CrossRefPubMedGoogle Scholar
  29. 29.
    Wu C, Zhao W, Yu J et al (2018) Induction of ferroptosis and mitochondrial dysfunction by oxidative stress in PC12 cells. Sci Rep 8:1–11. CrossRefGoogle Scholar
  30. 30.
    Hentze MW, Muckenthaler MU, Galy B, Camaschella C (2010) Two to tango: regulation of mammalian iron metabolism. Cell 142:24–38. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Zhao GY, Di DH, Wang B et al (2014) Iron regulates the expression of ferroportin 1 in the cultured hFOB 1.19 osteoblast cell line. Exp Ther Med 8:826–830. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Chen Y, Qian ZM, Du J et al (2005) Iron loading inhibits ferroportin1 expression in PC12 cells. Neurochem Int 47:507–513. CrossRefPubMedGoogle Scholar
  33. 33.
    Lane DJR, Richardson DR (2014) Free radical biology and medicine the active role of vitamin C in mammalian iron metabolism: much more than just enhanced iron absorption! Free Radic Biol Med 75:69–83. CrossRefPubMedGoogle Scholar
  34. 34.
    Muckenthaler MU, Rivella S, Hentze MW, Galy B (2017) A red carpet for iron metabolism. Cell 3:1–18. CrossRefGoogle Scholar
  35. 35.
    Thomsen MS, Andersen MV, Christoffersen PR et al (2015) Neurodegeneration with inflammation is accompanied by accumulation of iron and ferritin in microglia and neurons. Neurobiol Dis 81:108–118. CrossRefPubMedGoogle Scholar
  36. 36.
    Enculescu M, Metzendorf C, Sparla R et al (2017) Modelling systemic Iron regulation during dietary iron overload and acute inflammation: role of hepcidin-independent mechanisms. PLoS Comput Biol 13:e1005322. CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Sebastiani G, Wilkinson N, Pantopoulos K (2016) Pharmacological targeting of the hepcidin/ferroportin axis. Front Pharmacol 7:1–11. CrossRefGoogle Scholar
  38. 38.
    Brasselagnel C, Karim Z, Letteron P et al (2011) Intestinal DMT1 cotransporter is down-regulated by hepcidin via proteasome internalization and degradation. Gastroenterology 140:1261–1271. CrossRefGoogle Scholar
  39. 39.
    De Domenico I, Zhang TY, Koening CL et al (2010) Hepcidin mediates transcriptional changes that modulate acute cytokine-induced inflammatory responses in mice. J Clin Invest 120:2395–2405. CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Nairz M, Schleicher U, Schroll A et al (2013) Nitric oxide–mediated regulation of ferroportin-1 controls macrophage iron homeostasis and immune function in Salmonella infection. J Exp Med 210:855–873. CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Urrutia P, Aguirre P, Esparza A et al (2013) Inflammation alters the expression of DMT1, FPN1 and hepcidin, and it causes iron accumulation in central nervous system cells. J Neurochem 126:541–549. CrossRefPubMedGoogle Scholar
  42. 42.
    Raha AA, Vaishnav RA, Friedland RP et al (2013) The systemic iron-regulatory proteins hepcidin and ferroportin are reduced in the brain in Alzheimer’s disease. Acta Neuropathol Commun 1:55. CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Urrutia PHE (2017) Hepcidin attenuates amyloid beta-induced inflammatory and pro-oxidant responses in astrocytes and microglia. J Neurochem 142:140–152. CrossRefPubMedGoogle Scholar
  44. 44.
    Chaston T, Chung B, Mascarenhas M et al (2008) Evidence for differential effects of hepcidin in macrophages and intestinal epithelial cells. Gut 57:374–382. CrossRefPubMedGoogle Scholar
  45. 45.
    Yin X, Wu Q, Monga J et al (2017) HDAC1 governs iron homeostasis independent of histone deacetylation in iron-overload murine models. Antioxid Redox Signal ars 2017:7161Google Scholar
  46. 46.
    Bardai FH, Price V, Zaayman M et al (2012) Histone deacetylase-1 (HDAC1) is a molecular switch between neuronal survival and death. J Biol Chem 287:35444–35453. CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Kohli RM, Zhang Y (2013) TET enzymes, TDG and the dynamics of DNA demethylation. Nature 502:472–479. CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Evstatiev R, Gasche C (2012) Iron sensing and signalling. Gut 61:933–952. CrossRefPubMedGoogle Scholar
  49. 49.
    Anderson ER, Taylor M, Xue X et al (2013) Intestinal HIF2α promotes tissue-iron accumulation in disorders of iron overload with anemia. Proc Natl Acad Sci U S A 110:E4922–E4930. CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Sanchez M, Galy B, Muckenthaler MU, Hentze MW (2007) Iron-regulatory proteins limit hypoxia-inducible factor-2α expression in iron deficiency. Nat Struct Mol Biol 14:420–426. CrossRefPubMedGoogle Scholar
  51. 51.
    Aydemir F, Jenkitkasemwong S, Gulec S, Knutson MD (2009) Iron loading increases ferroportin heterogeneous nuclear RNA and mRNA levels in murine J774 macrophages. J Nutr 139:434–438. CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Harada N, Kanayama M, Maruyama A et al (2011) Nrf2 regulates ferroportin 1-mediated iron efflux and counteracts lipopolysaccharide-induced ferroportin 1 mRNA suppression in macrophages. Arch Biochem Biophys 508:101–109. CrossRefPubMedGoogle Scholar
  53. 53.
    Taylor M, Qu A, Anderson ER et al (2011) Hypoxia-inducible factor-2α mediates the adaptive increase of intestinal ferroportin during iron deficiency in mice. Gastroenterology 140:2044–2055. CrossRefPubMedPubMedCentralGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Laboratory of Neurobiology, Biomedicine Group, Department of Health Science and TechnologyAalborg UniversityAalborg EastDenmark

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