uPA binding to PAI-1 induces corneal myofibroblast differentiation on vitronectin.
OBJECTIVE
Vitronectin (VN) in provisional extracellular matrix (ECM) promotes cell migration. Fibrotic ECM also includes VN and, paradoxically, strongly adherent myofibroblasts (Mfs). Because fibrotic Mfs secrete elevated amounts of urokinase plasminogen activator (uPA), we tested whether increased extracellular uPA promotes the persistence of Mfs on VN.


METHODS
Primary human corneal fibroblasts (HCFs) were cultured in supplemented serum-free medium on VN or collagen (CL) with 1 ng/mL transforming growth factor β1 (TGFβ1). Adherent cells were quantified using crystal violet. Protein expression was measured by Western blotting and flow cytometry. Transfection of short interfering RNAs was performed by nucleofection. Mfs were identified by α-smooth muscle actin (α-SMA) stress fibers. Plasminogen activator inhibitor (PAI-1) levels were quantified by ELISA.


RESULTS
TGFβ1-treated HCFs secreted PAI-1 (0.5 uM) that bound to VN, competing with αvβ3/αvβ5 integrin/VN binding, thus promoting cell detachment from VN. However, addition of uPA to cells on VN increased Mf differentiation (9.7-fold), cell-adhesion (2.2-fold), and binding by the VN integrins αvβ3 and -β5 (2.2-fold). Plasmin activity was not involved in promoting these changes, as treatment with the plasmin inhibitor aprotinin had no effect. A dominant negative PAI-1 mutant (PAI-1R) that binds to VN but does not inhibit uPA prevented the increase in uPA-stimulated cell adhesion and reduced uPA-stimulated integrin αvβ3/αvβ5 binding to VN by 73%.


CONCLUSIONS
uPA induction of TGFβ1-dependent Mf differentiation on VN supports the hypothesis that elevated secretion of uPA in fibrotic tissue may promote cell adhesion and the persistence of Mfs. By blocking uPA-stimulated cell adhesion, PAI-1R may be a useful agent in combating corneal scarring.
Department of Ophthalmology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Lingyan
L
Wang
Christine M
CM
Ly
Chun-Ying
CY
Ko
Erin E
EE
Meyers
Daniel A
DA
Lawrence
Audrey M
AM
Bernstein
D015153:Blotting, Western; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D058628:Myofibroblasts / Q000502:physiology*; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism / Q000502:physiology*; D034741:RNA, Small Interfering; D014162:Transfection; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000494:pharmacology*; D019096:Vitronectin / Q000494:pharmacology*
D017395:Plasminogen Activator Inhibitor 1; D034741:RNA, Small Interfering; D053773:Transforming Growth Factor beta1; D019096:Vitronectin; D014568:Urokinase-Type Plasminogen Activator