Ferritin and HKa-Mediated Signaling Figure 2. Ferritin reverses the anti-adhesive properties of HKa. Cells were incubated with HKa (50 nM) alone, HKa (50 nM) plus ferritin (100 nM), or ferritin (100 nM) alone and allowed to adhere on vitronectin-coated coverslips in the presence of 20 ng/ml bFGF and 10 mM ZnCl2 for two hours. A. Adherent cells were fixed and stained with crystal violet and visualized by microscopy; B. Adherent cells per 106 field were counted; shown are means and standard deviation of 3 experiments with * p,0.001 and ** p,0.0001. C. Adherent cells were lysed and analyzed by western blotting. D. Phosphorylation of signaling molecules was quantified by densitometry. Data is expressed as percent phosphorylated/total protein; shown are means and standard deviation of three independent experiments; *p,0.02; **p,0.003. doi:10.1371/journal.pone.0040030.g002 functional differences [9] and bind to different receptors [16,17,36]. Ferritin found in the plasma (serum ferritin) is composed predominantly of a subunit closely related to the L type [15]. To determine whether ferritin subunit composition affected its ability to antagonize HKa activity, we first tested whether recombinant ferritins bound to HKa in a pull-down experiment. For these experiments we used a protein in which domain 5 of HKa, which contains the HGK-rich peptide sequence, was fused to GST [20]. Recombinant ferritin homopolymers composed either solely of the L subunit or solely of the H subunit [37] were biotinylated, incubated with GST-tagged domain 5 of HKa, and immunoprecipitated with anti-GST antibody. As shown in Figure 4A, ferritins composed of either subunit associated with domain 5 of HKa (lanes 1–4). Controls indicated that GST alone did not associate with either HFt or LFt (Figure S2). We then tested whether HFt and LFt were equally capable of blocking the anti-proliferative effects of HKa on endothelial cells. As shown in Figure 4B, treatment with either HFt or LFt significantly blocked loss of viability in HKa-treated cells (p,0.004). To determine whether these protective effects of HFt and LFt were associated with restoration of adhesion or survival signaling, we treated endothelial cells with HKa in the presence or absence of HFt or LFt or the combination of HFt and LFt for either short or long term, and measured effects on adhesion and survival signaling, respectively. As shown in Figure 4C–D and Figure 5, both recombinant ferritins restored activation of paxillin and Erk in cells treated with HKa. Thus, ferritins composed of either subunit can restore adhesion and survival signaling in endothelial cells treated with HKa. Ferritin reduces binding of HKa to endothelial cells and restores association of uPAR with a5b1 integrin HKa initiates signaling pathways in endothelial cells by binding to specific cell surface receptors. The binding site for HKa on endothelial cells includes uPAR [38] and other proteins [39,40]. uPAR is a GPI-linked cell surface receptor that recruits other membrane proteins, including integrins a5b1, into signaling complexes, permitting the triggering of downstream events, such as activation of Erk and Akt [41]. To test whether ferritin blocks HKa signaling by diminishing binding of HKa to the cell surface, we used flow cytometry. As seen in Figure 6, fluorescently labeled PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e40030