Urokinase receptor (CD87) aggregation triggers phosphoinositide hydrolysis and intracellular calcium mobilization in mononuclear phagocytes.
Leukocytes utilize urokinase receptors (uPAR; CD87) in adhesion, migration, and matrix proteolysis. uPAR aggregate at cell-substratum interfaces and at leading edges of migrating cells, so this study was undertaken to determine whether uPAR aggregation is capable of initiating activation signaling. Monocyte-like U937 cells were labeled with fluo-3-acetoxymethyl ester to quantitate intracellular Ca2+ concentrations ([Ca2+]i) by spectrofluorometry, and uPAR was aggregated by mAb cross-linking. uPAR aggregation induced highly reproducible increases in [Ca2+]i of 103.0 +/- 10.9 nM (p < 0.0001) and >3-fold increases in cellular d-myoinositol 1,4,5-trisphosphate (Ins(1,4,5)P3) levels. Similar increases in [Ca2+]i were also elicited by uPAR aggregation in human monocytes, but cross-linking a control IgG2a had no effect on [Ca2+]i. Selectively cross-linking uPA-occupied uPAR with an anti-uPA mAb produced smaller increases in [Ca2+]i, but fully saturating uPAR with exogenous uPA enhanced the [Ca2+]i response to equal the effect of aggregating uPAR directly. Increased [Ca2+]i was inhibited by thapsigargin, herbimycin A, and U73122, but only partially reduced by low extracellular [Ca2+], indicating that uPAR aggregation increases [Ca2+]i by activating phospholipase C through a tyrosine kinase-dependent mechanism, generating Ins(1,4,5)P3 and releasing Ca2+ from Ins(1,4, 5)P3-sensitive intracellular stores. Cross-linking the beta2 integrin CR3 could not duplicate the effect of uPAR cross-linking, and uPAR-triggered Ca2+ mobilization was not blocked by anti-CR3 mAbs. These results indicate that uPAR aggregation initiates phosphoinositide hydrolysis by mechanisms that are not strictly dependent on associated uPA or CR3.
Sitrin
R G
RG
Pulmonary and Critical Care Medicine Division, Hematology/Oncology Division, Department of Internal Medicine, and Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI 48109, USA. rsitrin@umich.edu
Pan
P M
PM
Harper
H A
HA
Blackwood
R A
RA
Todd
R F
RF
D016227:Benzoquinones; D020013:Calcium Signaling; D002448:Cell Adhesion; D002465:Cell Movement; D004963:Estrenes / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D007152:Immunologic Capping; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D047029:Lactams, Macrocyclic; D007975:Leukotriene B4 / Q000494:pharmacology; D016177:Macrophage-1 Antigen / Q000378:metabolism; D009000:Monocytes / Q000502:physiology*; D010716:Phosphatidylinositols / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology; D011809:Quinones / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D017828:Rifabutin / Q000031:analogs & derivatives; D019284:Thapsigargin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism; D020298:U937 Cells
D016227:Benzoquinones; D004963:Estrenes; D047029:Lactams, Macrocyclic; D016177:Macrophage-1 Antigen; C524898:PLAUR protein, human; D010716:Phosphatidylinositols; D011760:Pyrrolidinones; D011809:Quinones; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D007975:Leukotriene B4; D017828:Rifabutin; D019284:Thapsigargin; C020754:herbimycin; D015544:Inositol 1,4,5-Trisphosphate; D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases