Ferritin and HKa-Mediated Signaling Figure 5. Recombinant HFt and LFt antagonize effects of HKa on adhesion signaling. Cells were suspended in basal medium containing 20 ng/ml bFGF and 10 m M ZnCl2 and allowed to adhere onto vitronectin-coated dishes for 2 hours in the presence of HKa (50 nM), recombinant HFt or LFt(100 nM), or the combination of HKa plus ferritin. A. Adherent cells were lysed and analyzed by western blotting. B. Phosphorylation was quantified by densitometry; shown are means and standard deviation of 3 independent experiments. *p,0.02; **p,0.004. doi:10.1371/journal.pone.0040030.g005 survival kinases. Erk signaling is critical to endothelial cell survival and sprouting in vitro [26,27] and in vivo [29]; Akt also plays a key role in the endothelial cell response to growth factors [28]. Both of these pathways are targeted by HKa [31,42]. The ability of ferritin to interfere with HKa-mediated inhibition of these pathways was associated with its ability to promote endothelial cell survival and proliferation (Figure 1, 3). HKa also inhibits endothelial cell adhesion to vitronectin by reducing phosphorylation of FAK and paxillin [24]. We found that in addition to its effects on survival, ferritin blocked inhibitory effects of HKa on adhesion signaling by interfering with HKa-dependent blockade of paxillin and FAK. Ferritin alone exerted no measurable effects on these pathways (Figures 1, 2, 3, 4). These observations may provide a molecular explanation for our findings that ferritin inhibits the antiprolifer- ative and apoptotic effects of HKa on endothelial cells and blocks the anti-angiogenic effect of HKa in an aortic ring angiogenesis assay (Figure 1) and the tumor environment [20]. We further identify uPAR as a key upstream node of ferritin and HKa interaction. Erk, Akt, FAK and paxillin are all downstream of uPAR, suggesting that interference with binding of HKa to uPAR and restoration of uPAR signaling are the proximate events in ferritin-mediated inhibition of HKa activity, as depicted in the model shown in Figure 7D. Supporting this interpretation, we observed that ferritin interacts directly with HKa (Figure 4) but not uPAR (Figure 7), and that ferritin reduces overall binding of HKa to cells (Figure 6). Critically, ferritin restored the association of integrin a5b1 with uPAR in cells treated with HKa (Figure 7A, B), an event that was associated with activation of Erk, paxillin and FAK (Figure 2). We suggest that collectively, these events act in concert to restore adhesion (Figure 2) and proliferation (Figure 3) in endothelial cells challenged with HKa. We demonstrate that the HGK-rich domain of HKa is critical to the ability of ferritin to modulate the biological activities of HKa. We previously showed in plate-binding assays that the binding of HKa to ferritin is mediated through a small HGK-rich region of HKa that maps to domain 5 [20]. Supporting previous observations [23], this domain was able to inhibit endothelial cell proliferation and adhesion signaling (Figure 3). We further observed that ferritin was able to counteract effects mediated by this HGK-rich peptide as well as the intact HKa protein (Figure 3). PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e40030