and human [17] cells, and has been reported to exert a pro- inflammatory effect on hepatic stellate cells [18]. Our laboratory identified ferritin as a protein that regulates the activity of HKa [19–21]. In particular, ferritin blocks the anti- angiogenic effects of HKa on endothelial cells both in vitro and in vivo [20]. HKa exerts its anti-angiogenic effect by inhibiting endothelial cell migration and proliferation, as well as by inducing apoptosis [20,22–24]. Ferritin antagonizes the ability of HKa to both induce apoptosis and inhibit the migration of cultured endothelial cells [20]. Ferritin is an effective HKa antagonist in vivo, blocking the inhibitory effect of HKa on angiogenesis in a tumor xenograft and restoring angiogenesis to control levels [20]. Ferritin also inhibits the production of HKa by inhibiting the proteolytic cleavage of its precursor, HK [19,21]. The mechanism by which ferritin antagonizes HKa is unknown. Ferritin binds to HKa; however, whether this underlies the ability of ferritin to block the anti-angiogenic effect of HKa on endothelial cells has not been investigated. Effects of ferritin on signaling pathways modulated by HKa are also unknown. In this manuscript, we explore the mechanisms underlying the antago- nistic effects of ferritin on HKa activity. We find that ferritin antagonizes HKa-mediated anti-angiogenic signaling at two key steps, blocking inhibitory effects of HKa on both survival and adhesion signaling. The interplay between ferritin and HKa, two endogenous proteins, may be important in determining angiogenic outcome in pathologic as well as physiologic settings. Results Ferritin-mediated enhancement of endothelial cell survival in the presence of HKa is associated with activation of Paxillin, AKT, and Erk We have previously shown that HKa inhibits endothelial cell survival in vitro and in vivo, and that ferritin antagonizes the antiproliferative activity of HKa [20]. Pro-survival effects of ferritin in HKa-treated cells are dose-dependent (Figure S1), and are observed using either metabolic [20] or clonogenic assays (Figure 1A). In vivo, ferritin counteracts the antiangiogenic effects of HKa in the tumor microenvironment [20]. In addition, as shown in Figure 1B, C ferritin blocks the inhibitory effects of HKa in an aortic ring assay, which measures angiogenic sprouting from normal blood vessels ex vivo [25]. These results suggest that ferritin may modulate the anti-angiogenic activity of HKa in a broad array of physiological and pathophysiological contexts. To elucidate the mechanism by which ferritin promotes cell survival and proliferation in endothelial cells exposed to HKa, we assessed effects of ferritin and HKa on survival signaling. In particular, we tested the ability of ferritin to modulate MAPK44/ 42 (Erk) and AKT, kinases that play central roles in governing cell survival and proliferation in numerous cell types, including endothelial cells [26–29]. We also examined effects of ferritin on paxillin, a downstream target of Erk that has been implicated in the apoptotic effects of HKa [30]. Endothelial cells were plated and allowed to adhere. To assess the role of ferritin in modulating the Erk pathway, in some cases cells were stimulated with bFGF or FXII. Cells were then treated with HKa in the presence or absence of ferritin and effects on signaling monitored 24 hours later using antibodies specific to activated Erk, AKT and paxillin. As shown in Figure 1D–E, phosphorylation of Erk, AKT and paxillin were all reduced when endothelial cells were treated with HKa. However, co-treatment with ferritin restored phosphorylation to levels seen in control cells that had not been treated with HKa. Effects of HKa and ferritin were both statistically significant (Figure 1E). Ferritin by itself did Ferritin and HKa-Mediated Signaling not affect any of these pathways (Figure 1). Ferritin did not restore phosphorylation of Erk in cells treated with the Erk inhibitor PD98059 (Fig. 1F), indicating that ferritin does not act as a non- specific stimulator of Erk activity. Similar results were obtained when Erk signaling was induced by treating endothelial cells with Factor XII [31]: HKa reduced phosphorylation of Erk and ferritin reversed this action (Fig. 1G). Collectively, these results demon- strate that ferritin restores Erk and AKT signaling in cells treated with HKa. Ferritin promotes endothelial cell adhesion and adhesion signaling in the presence of HKa We next tested whether ferritin might also contribute to endothelial cell survival by blocking the anti-adhesive activity of HKa, since HKa has been shown to block adhesion of endothelial cells to provisional extracellular matrix proteins (ECM) such as vitronectin [24]. To test effects of ferritin on adhesion, we first performed a short- term adhesion assay. HUVEC cells were plated on vitronectin- coated dishes in the presence or absence of bFGF. At the time of plating, cells were also treated with HKa alone, ferritin alone, or with the combination of ferritin and HKa. Untreated cells served as a control. Two hours post–plating and treatment, control cells were adherent. Non-adherent cells were removed from the surface by washing, and remaining adherent cells were fixed and stained with crystal violet and visualized by microscopy. As seen in Figure 2A and 2B, HKa significantly inhibited adhesion of cells to vitronectin; however, co-treatment with ferritin enabled the cells to adhere to vitronectin. Next, we tested whether ferritin-promoted adhesion was associated with restoration of signaling pathways disrupted by HKa. Cell adhesion and spreading are controlled by complex signaling events mediated by proteins of the cell surface and the extracellular matrix. Key kinases in these signaling events are FAK and Erk, which converge on the common downstream target paxillin, an adaptor protein whose phosphorylation is required for integrin-mediated cytoskeletal reorganization [32]. To test effects of ferritin on adhesion signaling, we measured its effect on FAK and Erk signaling under short-term treatment conditions. Endothelial cells were plated in the presence of bFGF with HKa alone, ferritin alone, or the combination of ferritin and HKa on vitronectin-coated plates. Controls were untreated. Cells were lysed two hours later and phosphorylation of FAK, paxillin and Erk were analyzed by western blot. Figure 2C and 2D demonstrates that in the presence of HKa alone, phosphorylation of paxillin and Erk diminished, despite the presence of bFGF. Co- treatment with HKa and ferritin restored phosphorylation of paxillin and Erk. Thus, restoration of short-term adhesion (Figure 2) as well as long-term survival (Figure 1) of HKa-treated endothelial cells by ferritin is associated with reestablishment of critical signaling pathways. Ferritin antagonizes the effects of HKa on endothelial cell viability and signaling by binding to an HGK-rich region of HKa HKa comprises five domains: amino-terminal domains 1–3 linked through a disulfide bond to domains 5 and 6 [33]. Within domain 5 lies a small 29 amino acid region termed the HGK-rich region (histidine-glycine-lysine-rich region from D474 to K502 [20]), that mediates number of key functions of HKa, including binding to the endothelial cell surface [34] and inhibition of adhesion, invasion, and metastasis [35]. We previously observed PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40030