Ferritin and HKa-Mediated Signaling Figure 1. Ferritin restores colony formation, angiogenesis, and phosphorylation of paxillin, Erk1/2, and AKT in HUVECs exposed to HKa. A. HUVECs were treated with 50 nM HKa with and without 100 nM ferritin in the presence of 20 ng/ml bFGF for 24 hours. Growth medium was replaced and colonies were allowed to grow for 10 days before fixing and staining with crystal violet. Means and standard deviations of 3 independent experiments are shown with *p,0.01; **p,0.002. B. Aortic rings were stimulated with 30 ng/ml VEGF and treated with 100 nM HKa alone or in combination with 200 nM ferritin for 48 hours. Angiogenic sprouts were photographed on day 5. C. The number of sprouts were quantified from three different rings for each condition with *p,0.002. D. HUVECs were treated with 50 nM HKa alone, 100 nM ferritin alone, or co- treated with HKa and ferritin in basal media containing 20 ng/ml bFGF and 10 mM ZnCl2 for 24 hours. Activation of paxillin, Erk and Akt were determined by western blotting using antibodies to phosphorylated (P) and total (T) proteins. E. Band intensities were quantified by densitometry using ImageJ. Means and standard deviations of 3 independent experiments are shown, *p,0.02; **p,0.0003. F. HUVECs were stimulated as in D, and treated with 100 mM PD98059 in presence and absence of 100 nM ferritin. G. Cells were stimulated with 62 nM FXII and treated as described in D. doi:10.1371/journal.pone.0040030.g001 that ferritin binds to this HGK-rich region of HKa in a cell-free system [21]. We therefore explored whether interaction of ferritin with the HGK-rich domain of HKa is responsible for ferritin’s ability to block anti-angiogenic effects of HKa on endothelial cells. We first assessed whether anti-proliferative effects of the HGK-rich D474- K502 domain of HKa could be inhibited by ferritin. Endothelial cells were incubated with HKa or the 29 amino acid HGK-rich domain of HKa in the presence or absence of ferritin. Consistent with previous results [20], HKa inhibited endothelial cell proliferation, and this effect was blocked by ferritin (Figure 3A). Stimulation with growth factors was not able to overcome the inhibitory effect of HKa or modulate ferritin’s ability to antagonize HKa (Figure 3B). We then examined if the HGK domain would exert a similar inhibitory effect as HKa on endothelial cells, and if ferritin would block these effects. As shown in Figure 3C, domain 5 of HKa reduced cell viability to 56%60.3% of control, and co- treatment with ferritin increased cell viability to 8564.0% of control (p,0.0004). The HGK-rich peptide exerted a similar effect, reducing viability 66%62.0, whereas a control peptide (G440-L473, corresponding to a 33 amino acid region of HKa located immediately adjacent to the HGK rich region) was not able to affect proliferation (Figure 3C). Co-treatment with ferritin blocked the inhibitory effect of the HGK peptide, but did not affect the response to the control peptide. Thus, ferritin exerts its pro-survival effects on endothelial cells by targeting a small HGK- rich region within domain 5 of HKa. To test whether the inhibitory effects of ferritin on HKa signaling mapped to the same HGK-rich domain of HKa, we treated cells with the 29 amino acid HGK-rich domain in the presence and absence of ferritin and assessed effects on adhesion signaling. As shown in Figure 3D and 3E, treatment of cells with the HGK rich domain inhibited activation of both Erk and paxillin. Ferritin alone had no effect on activation of these signaling molecules. However, co-treatment with ferritin blocked the inhibitory effect of the HGK rich domain on both phosphorylation of paxillin and Erk. These results demonstrate that ferritin restores adhesion signaling by interfering with the activity of the HGK rich domain of HKa. Recombinant human Ferritin-H and Ferritin-L bind to domain 5 of HKa Ferritin is composed of an admixture of 24 subunits of the H and L type. H and L subunits assemble in various ratios to create ‘‘H-rich’’ (HFt) or ‘‘L-rich’’ (LFt) molecules. The H and L subunits of ferritin exhibit some sequence similarity, but also exhibit PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e40030