Outside-In Signaling upon Assembly on Endothelial Cells of the Proteins of the Plasma Kallikrein/Kinin System.

Investigations from our laboratory have shown that there is a multiprotein receptor system on endothelial cells (HUVEC) consisting of gC1qR, uPAR, and cytokeratin 1 for high molecular weight kininogen (HK) and factor XII (XII) (Blood97:2342; 99:3585) that serves as a presentation receptor for prekallikrein (PK) activation. When PK binds to HK on HUVEC, it is activated to plasma kallikrein by the serine protease prolylcarboxypeptidase (JBC277:17962; Blood103:4554). HK is also known to have anti-proliferative and anti-angiogenic activity. We asked if there is outside-in signaling through this HUVEC receptor complex. Initial investigations determined if single chain urokinase (ScuPA) or XII stimulates Erk 1 and 2 (MAPK42 and 44) (MAPK) in HUVEC. Independently ScuPA (5–200 nM) or XII (15–200 nM) in the presence of 0.05 mM zinc ion stimulates HUVEC MAPK expression and it is blocked by 0.1 mM PD98059, 30 nM wortmann, or 0.05 mM LY294002. Two chain uPA or APMSF-treated ScuPA upregulates MAPK to the same extent as ScuPA. Similarly, XIIa or APMSF-treated XII upregulates MAPK like XII. Since HK, ScuPA, XII and vitronectin (VN) all bind to the same region on uPAR (JBC279:16621), studies focused on the role of uPAR in these activities. Mab3B10 to the HK, ScuPA, XII, and VN binding site on uPAR’s Domain 2 (D2) blocks ScuPA or XII upregulation of MAPK. Peptides LRG20, YLP20, PGS20, or FHN20 from uPAR’s D2 (L166-N200) block ScuPA or XII upregulation of MAPK. Similarly, HKa (1 micromolar) and peptides from the HK Domain 5 (G469-H498) HUVEC binding region (GGH18, HKH20) (JBC270:19256) block ScuPA- or XII-induced MAPK expression. Treatment of HUVEC with 5-20 mM methyl-beta-cyclodextran (MBCD) or fillipin (1 microgram/ml), agents known to disrupt lipid rafts, do not interfere with ScuPA- or XII-induced MAPK upregulation. These combined data indicate that zymogen ScuPA or XII, two proteins with growth factor regions, directly signal through uPAR to upregulate MAPK to possibly induce cellular proliferative activities. HK or its activated fragments bound to uPAR inhibits these potential growth promoting activities. These studies imply that HKa’s anti-proliferative and anti-angiogenic activities are mediated through its interaction with uPAR. These investigations also indicate that, in addition to a presentation complex for PK activation, the HUVEC multiprotein receptor complex for HK, XII, and ScuPA has an auto-regulating outside-in signaling system in endothelials cells.