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. 2017 Jan 31;7:41770.
doi: 10.1038/srep41770.

The Fibrogenic Actions of Lung Fibroblast-Derived Urokinase: A Potential Drug Target in IPF

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Free PMC article

The Fibrogenic Actions of Lung Fibroblast-Derived Urokinase: A Potential Drug Target in IPF

Michael Schuliga et al. Sci Rep. .
Free PMC article

Abstract

The role of urokinase plasminogen activator (uPA) in idiopathic pulmonary fibrosis (IPF) remains unclear. uPA-generated plasmin has potent fibrogenic actions involving protease activated receptor-1 (PAR-1) and interleukin-6 (IL-6). Here we characterize uPA distribution or levels in lung tissue and sera from IPF patients to establish the mechanism of its fibrogenic actions on lung fibroblasts (LFs). uPA immunoreactivity was detected in regions of fibrosis including fibroblasts of lung tissue from IPF patients (n = 7). Serum uPA levels and activity were also higher in IPF patients (n = 18) than controls (n = 18) (P < 0.05), being negatively correlated with lung function as measured by forced vital capacity (FVC) %predicted (P < 0.05). The culture supernatants of LFs from IPF patients, as compared to controls, showed an increase in plasmin activity after plasminogen incubation (5-15 μg/mL), corresponding with increased levels of uPA and IL-6 (n = 5-6, P < 0.05). Plasminogen-induced increases in plasmin activity and IL-6 levels were attenuated by reducing uPA and/or PAR-1 expression by RNAi. Plasmin(ogen)-induced mitogenesis was also attenuated by targeting uPA, PAR-1 or IL-6. Our data shows uPA is formed in active regions of fibrosis in IPF lung and contributes to LF plasmin generation, IL-6 production and proliferation. Urokinase is a potential target for the treatment of lung fibrosis.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. uPA and IL-6 immunoreactive cells are detected in fibrotic lung tissue of IPF patients.
Serial sections of parenchymal tissue from separate IPF patients, (a–c) ALF016, (d–f) ALF023, (g–i) ALF028 and (j–l) ALF029, as well as a control donor, (m–o) ALF012. Sections were immunostained for uPA (left panel) or IL-6 (middle panel). The negative rabbit IgG control of the lung sections is also shown (right panel). The sections from IPF patients show uPA and IL-6 staining (brown) in fibroblast (Fb) and epithelial (Ep) cells. The scale bar in images are 100 micron.
Figure 2
Figure 2. uPA and α-SMA immunoreactivity overlap in fibrotic lung tissue of IPF patients.
Sequential serial lung tissue sections from either (ab) IPF patient (ALF027) or (cd) control (ALF024) stained for (a,c) uPA or (b,d) α-smooth muscle actin (α-SMA). The scale bars in images are (a–b) 200 or (c–d) 100 micron.
Figure 3
Figure 3. Serum levels of uPA are increased in IPF.
Sera (10 μg protein) from IPF or control donors in parallel were resolved by SDS-PAGE and immunoblotted for uPA. (a) Blots of serum samples showing overall protein loading by Ponceau red staining and both the high and low molecular weight forms of uPA (~54 and 31 kDa, respectively). Lane 1 is serum from a control donor, whereas lanes 2 and 3 are sera from separate IPF donors. (b) The relative levels of total uPA (high and low MW forms combined) in serum based on densitometry analysis. **P < 0.01 (n = 18 IPF and n = 18 non-IPF). (c) The percentage of low MW uPA to total uPA. *P < 0.05. (df) The relationships between uPA levels of IPF patients and lung function as measured by the % predicted values of FVC, FEV1 and DLCO. The solid line is the regression line, whereas the dotted lines represent the 95% confidence intervals. Data (df) were analyzed using the Pearson’s correlation test, with r and P (one-tailed) values provided.
Figure 4
Figure 4. Plasminogen stimulates increased IL-6 and uPA production by LFs in association with greater plasmin activity.
Levels of (a) plasmin activity, (b) uPA, (c) PAI-1 and (d) IL-6 in the media conditioned by LFs from IPF (n = 6) and control (n = 5–6) donors incubated with plasminogen for 24 h. *P < 0.05, **P < 0.01.
Figure 5
Figure 5. uPA and PAR-1 regulate the conversion of plasminogen into plasmin and subsequent IL-6 production.
(a) Levels of uPA in cultures of RNAi-transfected LFs. Data analyzed by student’s t test. (b) An immunoblot which shows PAR-1 protein knock-down in cell lysates by RNAi transfection (representative of 3 experiments). Supernatant levels of (c) plasmin activity and (d) IL-6 in cell cultures following RNAi transfection and 24 h plasminogen (15 μg/mL) incubation. *P < 0.05, **P < 0.01 (n = 3–5).
Figure 6
Figure 6. Plasmin(ogen) stimulates LF proliferation in an IL-6-dependent manner.
(a) Attached cell number following incubation with plasminogen (1.5–15 μg/mL) for 48 h (n = 5 control donors and n = 7 IPF donors). (b) Attached cell number following RNAi transfection and subsequent plasminogen (Plg, 15 μg/mL) or plasmin (Plm, 5 mU/mL) incubation. (c) Effect of SCH79797, a PAR-1 inhibitor, on plasmin-stimulated increases in cell number. (d) Cell number following incubation with plasmin and anti-IL-6 or control IgG (2 μg/mL) for 48 h. *P < 0.05, **P < 0.01, (n = 3–7).
Figure 7
Figure 7. uPA mediates increased IL-6 production in lung injury and disease.
uPA stimulates increased IL-6 production in lung fibroblasts by the regulation of plasmin (Plm) formation and activity, which perpetuates fibrosis in IPF.

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