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 establ ished stable transfectants of mouse erythroleukemia cells overexpressi ng the H-ferritin subunit. Analyses were performed on individual clone s of transfected cells induced to differentiate with hexamethylenbisac etamide (HMBA). The results showed that there was a reduction in the a mount of hemoglobin produced. in inverse relationship with the level o f H-subunit overexpression. Incorporation of [2-C-14]glycine into heme was reduced by 20% to 30% in the clones overexpressing H-ferritin sub unit compared with control clone. However, the reduction in hemoglobin production was not reversed by addition of heme precursors (delta-ami nolevulinic acid or iron) or by hemin itself. A reduced accumulation o f beta-globin mRNA was also observed. which could account for the impa ired hemoglobin synthesis. Furthermore, synthesis of the endogenous L- ferritin subunit was greatly repressed. Gel retardation assays perform ed on cytoplasmic extracts of transfected cells using an iron-responsi ve element (IRE) as a probe revealed that in overexpressing cells, the iron-regulatory protein (IRP) had a conformation with a high RNA-bind ing affinity, thus leading to translational repression of the endogeno us L-ferritin synthesis. These data suggest that an increased formatio n 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 fo r the role of IRP-mediated regulation of ferritin expression in erythr oid cell metabolism. (C) 1996 by The American Society of Hematology.