Characterization of non-transferrin (non-Tf) iron transport by K562 cells has revealed unique properties relative to iron uptake mechanisms present in other cell types (Inman, R. S., and Wessling-Resnick, M. (1993) J. Biol. Chem. 268, 8521-8528). Since treatment of K562 cells with phorbol esters promotes stable megakaryocytic differentiation, we examined the uptake of non-Tf iron in response to protein kinase C activation. Treatment of K562 cells with phorbol esters increased the cellular

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... of 55 Fe 4 -6-fold compared with untreated cells. The phorbol ester-induced stimulation of 55 Fe uptake was time-and dose-dependent, with significantly enhanced transport observed only after prolonged administration of 50 nM phorbol 12,13-dibutyrate (>8 h). These effects can be attributed to an increased V max of transport (14.0 ؎ 5 versus 0.6 ؎ 0.2 pmol/min/10 6 cells) as well as a 6-fold increase in the apparent K m (1.2 ؎ 0.4 versus 0.2 ؎ 0.06 M). It is thought that the reduction of Fe 3؉ to Fe 2؉ is required as a first step in the uptake mechanism, and the associated ferrireductase activity of K562 cells is also enhanced with phorbol ester treatment by 5-10-fold (337 ؎ 53 versus 43 ؎ 3 pmol/min/ 10 6 cells). Bryostatin-1, a protein kinase C activator that fails to induce differentiation of K562 cells, did not promote this effect, indicating that the enhanced transport activity is dependent on the differentiation response. The idea that synthesis of a new class of transporters is responsible for this effect is supported by the observation that actinomycin D blocks up-regulation of non-Tf iron transport. The increased transport and ferrireductase activity induced upon differentiation also correlate with the appearance of saturable iron-binding sites on the surface of K562 cells with K d ϳ 0.4 M. These results indicate that non-Tf iron transport activity and the expression of cell-surface iron-binding proteins can be controlled by environmental factors that promote megakaryocytic differentiation of K562 cells. Although the major pathway for the cellular delivery of iron is via receptor-mediated endocytosis of transferrin (Tf), 1 considerable evidence for alternative pathways of non-Tf iron uptake has been established. Properties defined for Tf-independ-