Iron-independent induction of ferritin H chain by tumor necrosis factor

Iron increases the synthesis of the iron-storage protein, ferritin, largely by promoting translation of preexisting mRNAs for both the H and L ferritin isoforms (H, heavy, heart, acidic; L, light, liver, basic). We have recently cloned and sequenced a full-length cDNA to murine ferritin H and identified ferritin H as a gene induced by tumor necrosis factor alpha (TNF-alpha, cachectin). Using primary human myoblasts, we have now examined the relationship between TNF-alpha and iron in regulating ferritin. Four lines of evidence suggest that TNF-alpha regulates ferritin independently of iron. First, evaluation of mRNA showed that TNF-alpha increased ferritin H chain specifically, provoking no change in steady-state levels of ferritin L mRNA; iron, in contrast, increased the mRNA of both isoforms. Second, the increase in ferritin H protein synthesis observed during TNF-alpha treatment was dependent on an increase in ferritin H mRNA: actinomycin D blocked the TNF-alpha-induced changes in ferritin H but did not inhibit the translational induction of ferritin seen with iron treatment. Third, equal ferritin mRNA induction was observed in iron-loaded cells and in cells depleted of iron by a permeant chelator, 2,2'-dipyridyl. Fourth, ferritin H induction by TNF-alpha and iron was additive over the entire range of iron concentrations, even at TNF-alpha doses known to maximally stimulate ferritin H mRNA levels. Nonetheless, the role of iron in translational regulation of ferritin was retained in TNF-alpha-treated cells; effective biosynthesis of TNF-alpha-induced, H-subunit-predominant ferritin protein required iron and could be enhanced by treatment of the cells with additional iron or blocked by 2,2'-dipyridyl. Finally, we observed that the TNF-alpha-mediated increase in ferritin synthesis peaked at 8 hr and was followed by a decrease in both H and L isoferritin synthesis; the addition of iron, however, reversed the late-occurring depression in ferritin synthesis. This suggests that TNF-alpha-induced synthesis of H-rich ferritin may reduce the regulatory pool of intracellular iron, secondarily inhibiting iron-mediated translation of ferritin mRNA. We conclude that TNF-alpha acts independently of iron in its induction of ferritin H mRNA but requires the presence of iron for this effect to be fully expressed at the protein level.