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-ferrrtin subunit. Analyses were performed on individual clones of transfected cells induced to differentiate with hexamethylenbisacet-amide (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% to 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 (5-aminolevulinic acid or iron) or by hemin itself. A reduced accumulation of β-globin mRIMA was also observed, which could account for the impaired hemoglobin synthesis. Furthermore, synthesis of the endogenous L-ferri-tin 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 β-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.