Ferritin and HKa-Mediated Signaling Figure 6. Ferritin inhibits binding of HKa to HUVECs. A–E. HUVEC cells were treated with 50 nM Alexa-Fluor 488 labeled HKa 6100 nM unlabeled spleen ferritin or BSA for one hour at 37uC. Cells were analyzed on FACSCaliber using CellQuest Pro Software. F. Means and standard deviation of 3 independent experiments. doi:10.1371/journal.pone.0040030.g006 Binding of ferritin to HKa is not ferritin subunit-specific. We observed that domain 5 of HKa interacted with ferritins composed of either the H or the L ferritin subunit types (Figure 4). Further, both HFt and LFt were able to counteract inhibitory effects of HKa on survival (Figure 4) and adhesive signaling (Figure 5). Although subunit composition varies by tissue type, natural ferritins generally contain some mixture of ferritin H and L subunits. These results suggest that all ferritins, independent of tissue source, are capable of exerting an inhibitory effect on HKa signaling. Human ferritin H and ferritin L have 56% amino acid sequence identity and similar overall structures, both in the monomeric and icositetrahedron form [43,44]. The ability of both ferritin H and L ferritin subunit types to interact with HKa suggests that binding of HKa to ferritin is mediated by a structural motif or sequence that is shared by the ferritin H and L subunits. The physiological source of ferritin that binds to HKa is not known. Circulating plasma ferritin is one possibility, and levels of this protein are roughly comparable to endogenous levels of HKa [21]. Because ferritin increases in inflammation or malignancy, plasma ferritin might be particularly effective at inhibiting HKa activity during these conditions. Further, because HKa is produced following docking and cleavage of HK on endothelial cells, locally produced HKa may recruit ferritin from the circulation to the endothelial cell surface. Alternatively or additionally, macrophages, which have recently been identified as a source of secreted ferritin [15], could serve as a local source of ferritin; concentrations of ferritin immediately adjacent to such cells may be appreciable, enabling ferritin to serve as an endogenous regulator of HKa that facilitates maintenance of endothelial cells. The ability of ferritin to bind to and inhibit the activity of HKa is a non-canonical ferritin function. Ferritin is principally known for its role in iron storage [9]. However, our previous work indicated that holoferritin (containing an iron core) and apoferritin (without iron) exhibited similar capacities to inhibit endothelial cell proliferation [20]. Our current observation that the ferritin subunits, which differ dramatically in their ferroxidase activity, are both able to bind to and inhibit HKa activity further suggests that the ability of ferritin to interact with HKa is independent of its iron storage function. This interpretation is congruent with the evolving view that ferritin may have extracellular signaling activities [18] in addition to its classical role in iron storage. Materials and Methods Cell culture Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Lonza (Walkersville, MD) and cultured in EGM-2 growth medium supplemented with 2% fetal bovine serum at 37uC in a humidified incubator at 5% CO2. Cells were used at passages 3–8. Cell treatment and viability assays Cell viability was assayed and quantified using 3-(4,5- Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent from Sigma. Cells were seeded in growth medium at a density of 6,000 cells per/well in a 96-well plate, allowed to adhere for 4 hours and the medium was replaced with M199 (Gibco) basal medium without growth factors overnight. Cells were treated in the presence or absence of 20 ng/ml of basic fibroblast growth PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e40030