Degradation of Internalized αvβ5 Integrin Is Controlled by uPAR Bound uPA: Effect on β1 Integrin Activity and α-SMA Stress Fiber Assembly

Myofibroblasts (Mfs) that persist in a healing wound promote extracellular matrix (ECM) accumulation and excessive tissue contraction. Increased levels of integrin αvβ5 promote the Mf phenotype and other fibrotic markers. Previously we reported that maintaining uPA (urokinase plasminogen activator) bound to its cell-surface receptor, uPAR prevented TGFβ-induced Mf differentiation. We now demonstrate that uPA/uPAR controls integrin β5 protein levels and in turn, the Mf phenotype. When cell-surface uPA was increased, integrin β5 levels were reduced (61%). In contrast, when uPA/uPAR was silenced, integrin β5 total and cell-surface levels were increased (2–4 fold). Integrin β5 accumulation resulted from a significant decrease in β5 ubiquitination leading to a decrease in the degradation rate of internalized β5. uPA-silencing also induced α-SMA stress fiber organization in cells that were seeded on collagen, increased cell area (1.7 fold), and increased integrin β1 binding to the collagen matrix, with reduced activation of β1. Elevated cell-surface integrin β5 was necessary for these changes after uPA-silencing since blocking αvβ5 function reversed these effects. Our data support a novel mechanism by which downregulation of uPA/uPAR results in increased integrin αvβ5 cell-surface protein levels that regulate the activity of β1 integrins, promoting characteristics of the persistent Mf

Cell-surface integrin β1 binding to collagen is increased after uPA-silencing but β1 activation is decreased.

<p>(A) HCFs were transfected with uPA siRNA or control siRNA. After 24 hours HCF were crosslinked to collagen. After cells were removed with 0.1% SDS, matrix-bound cross-linked proteins were released and equal amounts of protein were analyzed for integrins β5 and β1 by Western blot. (B) Cell adhesion: HCFs were transfected with uPA siRNA. After 24 hours cells were detached and seeded with blocking antibody to β5, β1 or control IgG. After 1 hour, adherent cells were quantified. **p value<0.01. (C–F) HCFs were transfected with either uPA siRNA or control siRNA and cells were analyzed after 24 hours. (C) Cell-surface biotinylation for integrin β1.(D) RT-PCR for uPA and integrin β1, *p value<0.05 and ***p value<0.001. (E) Flow cytometry for integrin β1. (F) Flow cytometry for activated integrin β1. N = 3–5 for each experiment.</p

β1 integrin is activated by blocking integrin β5.

<p>(A) HCFs were transfected with uPA siRNA or control siRNA and seeded with a blocking αvβ5 antibody or matched IgG. After 24 hours HCF were crosslinked to collagen. The amount of β1 released from the matrix after cell removal is visualized by Western blot. (B) HCFs were transfected with uPA siRNA. After 24 hours, cells were analyzed by flow cytometry for activated integrin β1. N = 3 for each experiment.</p

Integrin β5 protein expression is increased on TGFβ-induced Mfs.

<p>HCFs were treated with TGFβ1 or FGF-2 plus heparin for 72 hours and analyzed for β5 and β3 cell-surface expression. (A) Flow cytometry (B) Cell-surface biotinylation; lysates were immunoprecipitated (IP) with antibody against integrin β5, separated by SDS-PAGE and probed with streptavidin-HRP to detect biotinylated proteins. Bands were identified by molecular weight. GAPDH indicates equal protein input for IP. Quantification of the β5 band relative to GAPDH input control is shown. (C) Real-time PCR for β5, β3, and uPAR at Day 1 and Day 7 after TGFβ1 treatment. *p value<0.05 for gene expression between Day 1 and Day 7 for each. (D) HCF were stimulated with TGFβ for 48 hours to initiate Mf differentiation and then treated with anti-αvβ5 function blocking antibodies or IgG controls overnight. Cells were fixed and immunostained for α-Smooth Muscle Actin (α-SMA). N = 3–5 for each experiment.</p

siRNA knockdown of uPA triggers incorporation of α-SMA into stress fibers.

<p>HCF were transfected with siRNA against uPA or control siRNA and after 24 hours (A) cells were immunostained for α-SMA. Bar = 10 um or 20 um. Images are from two independent experiments. (B) Metamorph analysis quantified changes in average cell size in each condition. ***p value<0.001. (C) Cell lysates were Western blotted for α-SMA and for uPA to confirm knock down. GAPDH indicates equal loading. Densitometry for α-SMA/GAPDH is shown. N = 3–5 for each experiment.</p

uPA expression controls integrin β5 levels.

<p>(A) HCFs were transfected with a non-cleavable uPAR mutant cDNA (uPAR NC) or control vector. After 48 hours, cells were lysed and subjected to Western blot analysis for integrin β5. GAPDH indicates equal loading. Cell associated PA activity was measured colorimetrically by adding plasminogen and a chromogenic substrate for plasmin to cell lysates. Cell associated PA activity is shown to the right. *p value<0.05. HCF were transfected with (B) uPA siRNA or non-targeting siRNA control or (C) uPAR siRNA or non-targeting siRNA control. After 24 hours, lysates were Western blotted for β5 and uPA or uPAR to confirm knockdown. (D) HCF were transfected with uPAR siRNA, uPA siRNA, or control siRNA. After 24 hours, lysates were Western blotted for uPA. The data in (D) demonstrate that uPA is still expressed after uPAR silencing with uPAR siRNA. GAPDH indicates equal loading. (E–G) HCFs were transfected with uPA siRNA or control siRNA. (E) Flow cytometry. (F) Cell-surface biotinylation: After 24 hours, HCFs were cell-surface biotinylated before lysing and IP with streptavidin-HRP beads followed by Western for β5. GAPDH indicates equal input of protein for IP. (G) Integrin β5 bound to vitronectin (VN), top: After 24 hours, HCFs were treated with a cleavable cross-linker and cells were removed by lysis with 0.1% SDS. Next, the cross-linker was cleaved, releasing VN-bound proteins. This fraction was concentrated equal protein was Western blotted for β5. Bottom: Cell adhesion on VN: 24 hours after transfection, cells were detached and plated on VN for another 24 hours. Cells were fixed, stained with DAPI and counted. **p value<0.01. N = 3–7 for each experiment.</p