Purpose
Previously, we demonstrated that Slurp1 (secreted Ly6/urokinase-type plasminogen activator receptor (uPAR)-related protein-1) is an abundantly expressed immunomodulatory protein in the cornea. Here, we examine the mechanisms by which Slurp1 functions.
Methods
Slurp1-interacting proteins were identified by ligand blots, wherein mouse kidney lysates were separated on native polyacrylamide gels, transferred to PVDF membranes, incubated with partially purified recombinant Slurp1, washed, and probed with anti-Slurp1 antibody. Slurp1 interaction with urokinase-type plasminogen activator (uPA) was confirmed by (i) re-probing the ligand blots with anti-uPA antibody, (ii) pull-down assays and (iii) enzyme-linked immunosorbent assays (ELISAs). Effect of Slurp1 on (i) cell proliferation, migration and interaction with extracellular matrix (ECM), and (ii) uPA interaction with cell surface-bound uPAR was examined by treating M/KT-1 cells with Slurp1, followed by proliferation, migration and binding assays, and immunofluorescent staining with anti-uPA and anti-uPAR antibody.
Results
Ligand blots revealed that Slurp1 binds many proteins in mouse kidney lysates. One of the Slurp1-interacting proteins in ligand blots co-migrated with the band corresponding to uPA. Pull-down assays and ELISAs confirmed Slurp1 interaction with uPA. Slurp1-treated M/KT-1 cells (i) proliferated at a slower rate, (ii) adhered with lower affinity to different ECM components, and (iii) migrated at a reduced pace. Immunofluorescent staining revealed that exogenous Slurp1 decreased the amount of cell surface-bound uPA.
Conclusions
Slurp1 efficiently interacts with the uPAR ligand uPA. Slurp1 suppresses the rate of cell proliferation, migration and the affinity of interaction with ECM by serving as a competing soluble scavenger of uPA.
Keywords: 480 cornea: basic science •
482 cornea: epithelium •
486 cornea: tears/tear film/dry eye