Overexpression of the ferritin H subunit in cultured erythroid cells changes the intracellular iron distribution.
To test the hypothesis that variations in H- and L-subunit composition in the ferritin shell affect intracellular iron metabolism, we established stable transfectants of mouse erythroleukemia cells overexpressing the H-ferritin subunit. Analyses were performed on individual clones of transfected cells induced to differentiate with hexamethylenbisacetamide (HMBA). The results showed that there was a reduction in the amount of hemoglobin produced, in inverse relationship with the level of H-subunit overexpression. Incorporation of [2-14C]glycine into heme was reduced by 20% t0 30% in the clones overexpressing H-ferritin subunit compared with control clone. However, the reduction in hemoglobin production was not reversed by addition of heme precursors (delta-aminolevulinic acid or iron) or by hemin itself. A reduced accumulation of beta-globin mRNA was also observed, which could account for the impaired hemoglobin synthesis. Furthermore, synthesis of the endogenous L-ferritin subunit was greatly repressed. Gel retardation assays performed on cytoplasmic extracts of transfected cells using an iron-responsive element (IRE) as a probe revealed that in overexpressing cells, the iron-regulatory protein (IRP) had a conformation with a high RNA-binding affinity, thus leading to translational repression of the endogenous L-ferritin synthesis. These data suggest that an increased formation of H-rich isoferritins leads to a rapid chelation of the regulatory iron pool. While the mechanism underlying the reduction in beta-globin mRNA remains to be elucidated, this study provides direct evidence for the role of IRP-mediated regulation of ferritin expression in erythroid cell metabolism.
Picard
V
V
INSERM U409, Génétique et Pathologie Moléculaires de l'Hématopoièse, Faculté Xavier Bichat, Paris, France.
Renaudie
F
F
Porcher
C
C
Hentze
M W
MW
Grandchamp
B
B
Beaumont
C
C
D000081:Acetamides / Q000494:pharmacology; D000622:Aminolevulinic Acid / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D015672:Erythroid Precursor Cells / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005293:Ferritins / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation; D005914:Globins / Q000096:biosynthesis / Q000235:genetics; D006418:Heme / Q000096:biosynthesis; D006427:Hemin / Q000494:pharmacology; D006454:Hemoglobins / Q000096:biosynthesis; D007424:Intracellular Fluid / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology; D051379:Mice; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection; D014407:Tumor Cells, Cultured
D000081:Acetamides; D006454:Hemoglobins; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D006418:Heme; D012313:RNA; D006427:Hemin; D000622:Aminolevulinic Acid; D005914:Globins; D005293:Ferritins; D007501:Iron; C014026:hexamethylene bisacetamide