Human retinal pigment epithelial lysis of extracellular matrix: functional urokinase plasminogen activator receptor, collagenase, and elastase.
OBJECTIVE
To show (1) human retinal pigment epithelial (HRPE) expression of functional urokinase plasminogen activator receptor (uPAR; CD87), (2) HRPE secretion of collagenase and elastase, (3) uPAR-dependent HRPE migration, and (4) uPAR expression in diseased human retinal tissue.


METHODS
Immunohistochemistry for uPAR was performed on cultured HRPE cells and in sections of human retina. Double-immunofluorescent staining of live human RPE cells with anti-CR3 antibody (CD11b) was performed to demonstrate the physical proximity of this beta 2 integrin with uPAR and determine whether associations were dependent on RPE confluence and polarity. Extracellular proteolysis by HRPE uPAR was evaluated using fluorescent bodipy-BSA and assessed for specificity by plasminogen activator inhibitor-1 (PAI-1) inhibition. The effect of interleukin-1 beta (IL-1 beta) on uPAR expression was assessed. Collagenase and elastase secretion by unstimulated and IL-1-stimulated HRPE cells was measured by 3H-labelled collagen and elastin cleavage. HRPE-associated collagenase was also assessed by cleavage of fluorescent DQ-collagen and inhibited by phenanthroline. Using an extracellular matrix assay, the roles of uPAR and collagenase in HRPE migration were assessed.


RESULTS
Immunoreactive uPAR was detected on cultured HRPE cells and increased by IL-1. On elongated, live HRPE cells, uPAR dissociated from CD11b (CR3) and translocated to anterior poles of migrating cells. Extracellular proteolysis was concentrated at sites of uPAR expression and specifically inhibited by PAI-1. Cultured HRPE cells secreted substantial, functional collagenase and elastase. IL-1 upregulated uPAR, collagenase, and elastase activities. Specific inhibition of uPAR, and to a lesser degree collagenase, reduced HRPE migration in matrix/gel assays. Immunoreactive uPAR was present along the HRPE basolateral membrane in retinal sections and in sections of diseased retinal tissue.


CONCLUSIONS
HRPE cells express functional uPAR, collagenase, and elastase, which may permit HRPE proteolysis and migration. uPAR polarization may concentrate proteolysis at the leading edge of migrating HRPE cells.
Elner
Susan G
SG
University of Michigan and University of Chicago, USA.
D002465:Cell Movement; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D017364:Collagenases / Q000378:metabolism*; D004549:Elastin / Q000378:metabolism; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D010196:Pancreatic Elastase / Q000378:metabolism*; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010960:Plasminogen Activators / Q000096:biosynthesis*; D011956:Receptors, Cell Surface / Q000096:biosynthesis*; D055293:Receptors, Urokinase Plasminogen Activator; D015854:Up-Regulation
D007375:Interleukin-1; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D003094:Collagen; D004549:Elastin; D010960:Plasminogen Activators; D010196:Pancreatic Elastase; D017364:Collagenases