uPA binding to PAI-1 induces corneal myofibroblast differentiation on vitronectin

Abstract

Purpose: 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.

Figure 1.

Adhesion and Mf differentiation of HCFs on VN was less than that on CL. (A) HCF adhesion on CL and VN. HCFs were seeded on CL- or VN-coated 96-well plates for 1 hour or 24 hours in the presence of TGFβ1 prior to fixation and detection with crystal violet. Adhesion on VN compared to CL was reduced at each time point. (B) HCFs were seeded on either CL or VN and treated with TGFβ1 for 72 hours prior to fixation and immunodetection for α-SMA containing stress fibers (red, arrow). The nucleus is stained with 4′,6-diamidino-2-phenylindole (blue). Bar = 100 μm. (C) The percentage of Mfs (HCFs with α-SMA containing stress fibers) on CL and VN were counted and graphed as percents of total cells. Mf formation on VN is significantly reduced. Standard errors of the means between experiments are shown. *P < 0.05, **P < 0.01, and ***P < 0.005. N = 3 for each experiment.

Figure 2.

uPA induced the organization of α-SMA containing stress fibers in HCFs on VN. (A) HCFs were seeded on VN and treated with TGFβ1 for 72 hours. Before fixation, cells were treated with or without 50 units/mL uPA for 3 hours and then fixed and immunodetected for α-SMA (red, arrow). The nucleus is stained with 4′,6-diamidino-2-phenylindole (blue). Bar = 100 μm. Images from two experiments are shown. Addition of uPA stimulated Mf differentiation on VN. (B) The percentage of Mfs under the two conditions were counted and graphed as a percentage of total cells. (C) α-SMA protein expression was similar in HCFs with or without uPA treatment. HCFs were treated as shown in (A) prior to lysis with radioimmunoassay radioimmunoprecipitation buffer and then Western blotted for α-SMA. GAPDH controls were used for equal loading. Addition of uPA did not affect α-SMA expression but did affect its incorporation into stress fibers. N = 3 for each experiment. **P < 0.01.

Figure 3.

uPA removed PAI-1 from VN. (A) HCFs were seeded on VN with TGFβ1 for 24 hours prior to treatment for 3 hours with or without uPA. Next, cells were detached, and, to detect PAI-1 associated with the matrix, the matrix was lysed in 1% SDS, immunoprecipitated for VN and Western blotted for PAI-1 (top). Detached cells were lysed in radioimmunoprecipitation assay (RIPA) buffer and Western blotted for PAI-1 and GAPDH (bottom). Treatment with uPA dissociated PAI-1 from VN. (B) To increase secreted uPA, HCFs were transfected with uPAR-targeted siRNA (siuPAR) or control siRNA (control) and seeded on VN in the presence of TGFβ1. After 24 hours, medium was collected for the detection of secreted uPA, cells were detached and lysed in RIPA and the matrix was lysed with 1% SDS. Samples were Western blotted for uPA, uPAR, and PAI-1. The increase in secreted uPA correlates with a decrease in PAI-1 on the matrix. N = 3 for each experiment. *P < 0.05, **P < 0.01, ***P < 0.005.

Figure 4.

uPA treatment induced the integrin αvβ3 and αvβ5 binding to VN. HCFs were seeded on VN with TGFβ1 for 24 hours prior to treatment with or without uPA for 3 hours. (A) HCFs were treated with a cleavable cross-linker, and cells were removed by lysis with 0.1% SDS. Next, the cross-linking was reversed, releasing protein bound to the matrix. This fraction was concentrated, and equal amounts of protein were Western blotted for detection of the integrin subunits β5 and β3 and for GAPDH. The absence of GAPDH demonstrates that cells had been removed prior to releasing the cross-linked fraction. Addition of uPA increased integrin binding to VN. (B) HCFs were lysed with radioimmunoprecipitation assay buffer and Western blotted for β3, β5, and GAPDH. GAPDH controls were used for equal loading. (C) Cell surface levels of integrins β3 and β5 were measured by flow cytometry. uPA treatment did not alter total or cell surface integrin expression. N = 3 to 5 for each experiment. *P < 0.05 and **P < 0.01.

Figure 5.

PAI-1R reduced cell adhesion on VN. (A) HCFS were seeded on VN with TGFβ1 for 24 hours prior to treatment with combinations of uPA, PAI-1R, or aprotinin for 3 hours, as shown, and then assayed for adhesion by crystal violet staining. Addition of uPA increased cell adhesion (compare lanes 1 and 2), while PAI-1R treatment reduced uPA-mediated effects (compare lanes 1 and 3). Aprotinin did not impact adhesion (compare lanes 1–4 to lanes 5–8). (B) Cross-linking assay for HCFs with uPA or PAI-1R treatment, as described in the legend to Fig. 4A. PAI-1R reduced the uPA-mediated increase in integrin binding to VN. (C) Cells were seeded on VN and incubated with 1 ng/mL TGFβ1 for 72 hours. Before fixation, cells were treated with uPA or uPA plus PAI-1R for 3 hours and then fixed and immunodetected for α-SMA (red). The nucleus was stained with 4′,6-diamidino-2-phenylindole (blue). Bar = 100 μm. (D) Cells treated as described in the legend to C were quantified α-SMA-stress fibers containing cells that were either spread or were narrow and detaching. PAI-1R reduced Mf spreading. N = 3 to 5 for each experiment. *P < 0.05, **P < 0.01, ***P < 0.005.