The Urokinase Plasminogen Activator Receptor Mediates ScuPA- or FXII-Induced Cell Growth and Proliferation through ERK1/2, Akt, and Beta-1-Integrin.
Authors: Schmaier Alvin H. , Mahdi Fakhri , Sitrin Robert 
Published: October 2019

Abstract
Previous studies have shown that the multiprotein kininogen (HK) and factor XII (FXII) receptor complex on endothelial cells (HUVEC) that consists of gC1qR, uPAR, and cytokeratin 1 (Blood97:2342, 99:3585) allows for prekallikrein assembly and activation for bradykinin formation (Blood 103:4554). New studies show outside-in signaling through this HUVEC receptor complex. Single chain urokinase (ScuPA) or FXII in the presence of 0.05 mM zinc ion stimulates ERK1/2 (MAPK42 and 44) in HUVEC (Blood 106:Abstract 1024Blood 106:Abstract 2005). Furthermore, cleaved HK (HKa) or peptides from the HK domain 5 cell binding region block HUVEC ERK1/2 phosphorylation. The region on uPAR that mediates this signaling is the same 22 aa sequence on uPAR domain 2 that binds ScuPA, FXII, HK, and vitronectin (JBC 279:16621). ScuPA or FXII phosphorylation of ERK1/2 is blocked by 0.1 mM PD98059, 30 nM wortmann, or 0.05 mM LY294002. Additional investigations determined if ScuPA or FXII in the presence of zinc ion stimulate Akt phosphorylation (Ser473). Both ScuPA (5–200 nM) or FXII (15–200 nM) in the presence of 0.05 mM zinc ion stimulated HUVEC Akt phosphorylation and it is blocked by 30 nM wortmann or 0.05 mM LY294002. Investigations next determined if the same region on uPAR that mediated ERK1/2 phosphorylation participated in Akt phosphorylation. Peptide LRG20 (L166-D185)or PGS20 (P176-T195) from domain 2 of uPAR or peptides HVL20 (H471-K494), HKH20 (H479-H498) or FKL20 (F459-K478) from domain 5 of HK blocked ScuPA- or FXII-induced phosphorylation of Akt (Ser473). Since uPAR does not directly interact with intracellular pathways, further investigations determined the mechanism for outside-in signaling. Studies showed that two antibodies to beta-1-integrin (Mabs 2253 and 1987) blocked ScuPA- or FXII-induced phosphorylation of Akt (Ser473). Additional investigations showed that ScuPA- (16–64 nM) or FXII- (60–240 nM) induced cell proliferation in a concentration-dependent fashion and it was blocked by peptide LRG20 of uPAR or HVL24 of HK’s domain 5. Further, cell proliferation is blocked by PD98059, wortmann, LY294002, and Mab 2253 to beta-1-integrin. Similarly, cell growth as measured by BrdU incorporation was induced by ScuPA or FXII in a concentration-dependent fashion. BrdU incorporation was blocked by peptides LRG20 and HVL24 as well as PD98059, wortmann, LY294002, and Mab 2253 to beta-1-integrin. These combined studies indicate uPAR in the kininogen multiprotein complex serves as at least one focal point for outside-in signaling after ScuPA or FXII binding. Stimulation of this receptor through beta-1-integrin leads to phosphorylation of ERK1/2 and Akt (Ser473) and induces HUVEC growth and proliferation. These investigations indicate two novel findings: first, they indicate outside-in signaling pathways for the multiprotein receptor complex for ScuPA, FXII, and HK. Second, they describe at least one pathway for the anti-angiogenic and anti-proliferative activities of HKa and its domain 5 fragments. These investigations show that the multiprotein kininogen/FXII receptor complex influences both HUVEC cellular growth and proliferation as well as intravascular proteolytic activity leading to bradykinin formation, blood pressure control and modulation of thrombosis risk (Blood 108:192).