Overexpression of wild type and mutated human ferritin H-chain in HeLa cells: in vivo role of ferritin ferroxidase activity.
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.
Cozzi
A
A
Dibit, Department of Biological and Technological Research, IRCCS H. San Raffaele, Milano, Italy.
Corsi
B
B
Levi
S
S
Santambrogio
P
P
Albertini
A
A
Arosio
P
P
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001052:Apoferritins; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D002570:Ceruloplasmin / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004318:Doxycycline / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005293:Ferritins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015151:Immunoblotting; D007501:Iron / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D018384:Oxidative Stress; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D013752:Tetracycline / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D000900:Anti-Bacterial Agents; D018076:DNA, Complementary; D005293:Ferritins; D001052:Apoferritins; D006861:Hydrogen Peroxide; D007501:Iron; D002570:Ceruloplasmin; D013752:Tetracycline; D004318:Doxycycline