Ferritin and HKa-Mediated Signaling Figure 4. Recombinant HFt and LFt bind to HKa and and inhibit its anti-proliferative activity. A. Binding of recombinant HFt and LFt to domain 5 of HKa. Purified and biotinylated recombinant HFt (20 mg) or LFt (20 mg) were incubated with 10 mg GST-D5 and the resulting complexes immunoprecipited with anti-GST antibody. Non-biotinylated HFt and LFt were used in the immunopreciptation shown in lane 4. The membranes were probed with streptavidin-HRP to detect biotinylated ferritin (b-ferritin) as well as with anti-GST antibody. B. Cells were treated with 50 nM HKa alone, or co-treated with 100 nM of HFt or LFt in the presence of 20 ng/ml bFGF and 10 mM ZnCl2. Cell viability was assessed using an MTT assay 24 hours post-treatment. Shown are means and standard deviation of triplicate experiments with ** p,0.004. C. Cell lysates were collected 24 hours post-treatment and analyzed by western blotting. D. Phosphorylation of signaling molecules was quantified by densitometry. Shown are means and standard deviation of 3 independent experiments. *p,0.04; **p,0.002. b = biotinylated. doi:10.1371/journal.pone.0040030.g004 were immunoprecipitated using anti-a5b1 integrin antibody and probed for the presence of uPAR using western blotting. As shown in Figure 7A, B, incubation with HKa dissociated uPAR from a5b1 integrin, consistent with previous reports [38]. However, co- treatment with ferritin restored complex formation between uPAR and integrin a5b1. Thus, incubation with ferritin prevents HKa from disrupting upstream (UPAR-mediated) as well as down- stream (Erk and Akt) signaling in endothelial cells. Ligand blotting demonstrates that ferritin binds to HKa but does not directly bind to uPAR (Figure 7C), suggesting that ferritin exerts its effects by displacing HKa from an association with uPAR rather than through direct interaction with uPAR. An overall model depicting effects of ferritin and HKa on endothelial cells is shown in Figure 7D. Discussion In this manuscript, we explore the mechanism underlying the cytoprotective effect of ferritin on endothelial cells exposed to HKa. We observe that (1) ferritin promotes adhesion of HKa- treated cells and restores critical survival and adhesion signaling pathways mediated by Erk, Akt, FAK and paxillin; (2) ferritin inhibits the anti-proliferative and anti-adhesive effects of HKa by targeting the HGK-rich domain of HKa; (3) both ferritins of the H and L subunit type can regulate HKa activity; and (4) ferritin diminishes binding of HKa to endothelial cells and restores the association of uPAR with a5b1 integrin. An overall model depicting effects of ferritin on HKa is shown in Figure 7D. Ferritin restores multiple signaling pathways disrupted by HKa. These include pathways mediated by Erk and Akt, key pro- PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e40030