Ferritin Blocks Inhibitory Effects of Two-Chain High Molecular Weight Kininogen (HKa) on Adhesion and Survival Signaling in Endothelial Cells Lia Tesfay1¤a, Annissa J. Huhn 2 , Heather Hatcher1, Frank M. Torti1,3¤a, Suzy V. Torti2,3*¤b 1 Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America, 2 Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America, 3 Comprehensive Cancer Center, Wake Forest School of Medicine, Winston-Salem, North Carolina, United States of America Abstract Angiogenesis is tightly regulated through complex crosstalk between pro- and anti-angiogenic signals. High molecular weight kininogen (HK) is an endogenous protein that is proteolytically cleaved in plasma and on endothelial cell surfaces to HKa, an anti-angiogenic protein. Ferritin binds to HKa and blocks its anti-angiogenic activity. Here, we explore mechanisms underlying the cytoprotective effect of ferritin in endothelial cells exposed to HKa. We observe that ferritin promotes adhesion and survival of HKa-treated cells and restores key survival and adhesion signaling pathways mediated by Erk, Akt, FAK and paxillin. We further elucidate structural motifs of both HKa and ferritin that are required for effects on endothelial cells. We identify an histidine-glycine-lysine (HGK) -rich antiproliferative region within domain 5 of HK as the target of ferritin, and demonstrate that both ferritin subunits of the H and L type regulate HKa activity. We further demonstrate that ferritin reduces binding of HKa to endothelial cells and restores the association of uPAR with a5b1 integrin. We propose that ferritin blocks the anti-angiogenic activity of HKa by reducing binding of HKa to UPAR and interfering with anti-adhesive and anti-proliferative signaling of HKa. Citation: Tesfay L, Huhn AJ, Hatcher H, Torti FM, Torti SV (2012) Ferritin Blocks Inhibitory Effects of Two-Chain High Molecular Weight Kininogen (HKa) on Adhesion and Survival Signaling in Endothelial Cells. PLoS ONE 7(7): e40030. doi:10.1371/journal.pone.0040030 Editor: Aamir Ahmad, Wayne State University School of Medicine, United States of America Received January 3, 2012; Accepted June 3, 2012; Published July 2, 2012 Copyright: 2012 Tesfay et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Institutes of Health (R01DK71892; R37 DK42421). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: storti@uchc.edu ¤a Current address: Department of Medicine, University of Connecticut Health Center, Farmington, Connecticut, United States of America ¤b Current address: Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut, United States of America Introduction Angiogenesis, the formation of new blood vessels from pre- existing vessels, is carefully regulated by a complex balance between pro- and anti-angiogenic signaling [1]. Disruption of this balance contributes to a number of pathological conditions. These include the excess angiogenesis seen in diabetic retinopathy [2], as well as the aberrant angiogenesis in the tumor microenvironment, which is believed to contribute to tumor progression and metastasis [3]. Conversely, defective angiogenesis can compromise wound healing [4]. High molecular weight kininogen (HK) is a plasma protein originally identified for its role in the intrinsic pathway of coagulation. HK is comprised of six domains, D1–D6. Endopro- teolytic cleavage of HK by kallikrein excises domain 4, yielding two bio-active molecules: bradykinin, contained within domain 4, and HKa, composed of domains 1–3 linked through a disulfide bond to domains 5 and 6 [5]. Bradykinin is a potent nonapeptide hormone with a 30 second half-life [6]. Among its other activities, bradykinin binds to G-coupled receptors and triggers NO release, which promotes angiogenesis. HKa, the other product formed from cleavage of HK, has a longer 9 hour half-life [7], and antagonizes bradykinin’s activity by serving as endogenous inhibitor of angiogenesis [8]. Proteins that modulate bradykinin or HKa activity therefore have the potential to exert an important effect on the balance between pro- and anti-angiogenic responses. Ferritin is a 24 subunit protein best known for its role in intracellular iron storage and detoxification (reviewed in [9]). Ferritin is composed of two subunit types, termed H and L, which share considerable sequence similarity. Twenty-four of these subunits assemble to form the mature ferritin protein. The H subunit contains a ferroxidase activity, important in iron oxidation, whereas the L subunit is important in iron nucleation, iron core formation, and protein stability. The ratio of H to L subunits is determined by tissue type and is also modulated by inflammatory and other stimuli. In addition to its intracellular localization, ferritin exists in the plasma, where it may serve roles in addition to its classic function in intracellular iron storage. For example, extracellular ferritin has been suggested to serve in iron delivery [10,11], and to exhibit immunosuppressive functions by affecting the proliferation and function of lymphocytes [10,12–14]. Extracellular ferritin appears to contain primarily L subunits, including a truncated version of ferritin L [15]. Serum ferritin is elevated not only in conditions of iron overload, but in acute and chronic inflammation and cancer. Extracellular ferritin binds to cell surface receptors on mouse [16] PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e40030