title: High molecular weight kininogen binds phosphatidylserine and opsonizes urokinase plasminogen activator receptor-mediated efferocytosis.
abstract: Phagocytosis of apoptotic cells (efferocytosis) is essential for regulation of immune responses and tissue homeostasis and is mediated by phagocytic receptors. In this study, we found that urokinase plasminogen activator receptor (uPAR) plays an important role in internalization of apoptotic cells and also characterized the underlying mechanisms. In a flow cytometry-based phagocytic assay, uPAR-deficient macrophages displayed significant defect in internalization but not tethering of apoptotic cells. When uPAR-deficient mice were challenged with apoptotic cells, they exhibited pronounced splenomegaly resulting from accumulation of abundant apoptotic cells in spleen. Overexpression of uPAR in HEK-293 cells enhanced efferocytosis, which was inhibited by Annexin V and phosphatidylserine (PS) liposome, suggesting that uPAR-mediated efferocytosis is dependent on PS. In serum lacking high m.w. kininogen (HK), a uPAR ligand, uPAR-mediated efferocytosis was significantly attenuated, which was rescued by replenishment of HK. As detected by flow cytometry, HK selectively bound to apoptotic cells, but not viable cells. In purified systems, HK was specifically associated with PS liposome. HK binding to apoptotic cells induced its rapid cleavage to the two-chain form of HK (HKa) and bradykinin. Both the H chain and L chain of HKa were associated with PS liposome and apoptotic cells. HKa has higher binding affinity than HK to uPAR. Overexpression of Rac1/N17 cDNA inhibited uPAR-mediated efferocytosis. HK plus PS liposome stimulated a complex formation of CrkII with p130Cas and Dock-180 and Rac1 activation in uPAR-293 cells, but not in control HEK-293 cells. Thus, uPAR mediates efferocytosis through HK interaction with PS on apoptotic cells and activation of the Rac1 pathway.
authors.affiliation: Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, First Affiliated Hospital, Soochow University, Suzhou 215006, China;
authors.forename: Aizhen
authors.initials: A
authors.lastname: Yang
authors.forename: Jihong
authors.initials: J
authors.lastname: Dai
authors.forename: Zhanli
authors.initials: Z
authors.lastname: Xie
authors.forename: Robert W
authors.initials: RW
authors.lastname: Colman
authors.forename: Qingyu
authors.initials: Q
authors.lastname: Wu
authors.forename: Raymond B
authors.initials: RB
authors.lastname: Birge
authors.forename: Yi
authors.initials: Y
authors.lastname: Wu
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D019679:Kininogen, High-Molecular-Weight / Q000378:metabolism; D008264:Macrophages / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010587:Phagocytosis / Q000502:physiology*; D010718:Phosphatidylserines / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D020349:Surface Plasmon Resonance; D014162:Transfection; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
chemical_list: D019679:Kininogen, High-Molecular-Weight; D010718:Phosphatidylserines; D055293:Receptors, Urokinase Plasminogen Activator; D020830:rac1 GTP-Binding Protein