Overexpression of wild type and mutated human ferritin H-chain in HeLa cells: in vivo role of ferritin ferroxidase activity

J Biol Chem. 2000 Aug 18;275(33):25122-9. doi: 10.1074/jbc.M003797200.

Abstract

Transfectant HeLa cells were generated that expressed human ferritin H-chain wild type and an H-chain mutant with inactivated ferroxidase activity under the control of the tetracycline-responsive promoter (Tet-off). The clones accumulated exogenous ferritins up to levels 14-16-fold over background, half of which were as H-chain homopolymers. This had no evident effect in the mutant ferritin clone, whereas it induced an iron-deficient phenotype in the H-ferritin wild type clone, manifested by approximately 5-fold increase of IRPs activity, approximately 2.5-fold increase of transferrin receptor, approximately 1.8-fold increase in iron-transferrin iron uptake, and approximately 50% reduction of labile iron pool. Overexpression of the H-ferritin, but not of the mutant ferritin, strongly reduced cell growth and increased resistance to H(2)O(2) toxicity, effects that were reverted by prolonged incubation in iron-supplemented medium. The results show that in HeLa cells H-ferritin regulates the metabolic iron pool with a mechanism dependent on the functionality of the ferroxidase centers, and this affects, in opposite directions, cellular growth and resistance to oxidative damage. This, and the finding that also in vivo H-chain homopolymers are much less efficient than the H/L heteropolymers in taking up iron, indicate that functional activity of H-ferritin in HeLa cells is that predicted from the in vitro data.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Bacterial Agents / pharmacology
  • Apoferritins
  • Cell Division / drug effects
  • Cell Division / genetics
  • Ceruloplasmin / metabolism
  • DNA, Complementary / metabolism
  • Doxycycline / pharmacology
  • Enzyme-Linked Immunosorbent Assay
  • Ferritins / chemistry*
  • Ferritins / genetics
  • Ferritins / metabolism*
  • HeLa Cells
  • Humans
  • Hydrogen Peroxide / pharmacology
  • Immunoblotting
  • Iron / metabolism
  • Mutagenesis
  • Mutation*
  • Oxidative Stress
  • Plasmids / metabolism
  • Precipitin Tests
  • Promoter Regions, Genetic
  • Tetracycline / metabolism
  • Time Factors
  • Transfection

Substances

  • Anti-Bacterial Agents
  • DNA, Complementary
  • Ferritins
  • Apoferritins
  • Hydrogen Peroxide
  • Iron
  • Ceruloplasmin
  • Tetracycline
  • Doxycycline