E4 engages uPAR and enolase-1 and activates urokinase to exert antifibrotic effects
Shailza Sharma, … , Roger A. Chambers, Carol Feghali-Bostwick
Shailza Sharma, … , Roger A. Chambers, Carol Feghali-Bostwick
Published December 22, 2021
Citation Information: JCI Insight. 2021;6(24):e144935. https://doi.org/10.1172/jci.insight.144935.
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Research Article Pulmonology Therapeutics

E4 engages uPAR and enolase-1 and activates urokinase to exert antifibrotic effects

Abstract

Fibroproliferative disorders such as systemic sclerosis (SSc) have no effective therapies and result in significant morbidity and mortality. We recently demonstrated that the C-terminal domain of endostatin, known as E4, prevented and reversed both dermal and pulmonary fibrosis. Our goal was to identify the mechanism by which E4 abrogates fibrosis and its cell surface binding partner(s). Our findings show that E4 activated the urokinase pathway and increased the urokinase plasminogen activator (uPA) to type 1 plasminogen activator inhibitor (PAI-1) ratio. In addition, E4 substantially increased MMP-1 and MMP-3 expression and activity. In vivo, E4 reversed bleomycin induction of PAI-1 and increased uPA activity. In patients with SSc, the uPA/PAI-1 ratio was decreased in both lung tissues and pulmonary fibroblasts compared with normal donors. Proteins bound to biotinylated-E4 were identified as enolase-1 (ENO) and uPA receptor (uPAR). The antifibrotic effects of E4 required uPAR. Further, ENO mediated the fibrotic effects of TGF-β1 and exerted TGF-β1–independent fibrotic effects. Our findings suggest that the antifibrotic effect of E4 is mediated, in part, by regulation of the urokinase pathway and induction of MMP-1 and MMP-3 levels and activity in a uPAR-dependent manner, thus promoting extracellular matrix degradation. Further, our findings identify a moonlighting function for the glycolytic enzyme ENO in fibrosis.

Authors

Shailza Sharma, Tomoya Watanabe, Tetsuya Nishimoto, Takahisa Takihara, Logan Mlakar, Xinh-Xinh Nguyen, Matthew Sanderson, Yunyun Su, Roger A. Chambers, Carol Feghali-Bostwick

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Figure 4

E4 peptide increases uPA and reduces PAI-1 levels in BALF and lung tissues.

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E4 peptide increases uPA and reduces PAI-1 levels in BALF and lung tissu...
PBS (n = 4), bleomycin (n = 5) (1.2 mU/g), or bleomycin and E4 (n = 5) (20 μg/mouse) was administered to C57BL/6J male mice. BALF and lung tissues were collected after 14 days. (A) Protein levels of uPA and PAI-1 were detected by immunoblotting (upper) and quantitative analysis (lower). (B) Activity levels of uPA and PAI-1 in BALF were measured by activity assays. (C) Protein levels of uPA and PAI-1 in mouse lung tissue homogenates were detected by immunoblotting. Western blots (upper) and quantitative analysis (lower) are shown. Samples were electrophoresed on the same gel in noncontiguous lanes. Loss of uPAR abrogates the antifibrotic effects of E4. (D) Normal lung fibroblasts were transfected with control or uPAR siRNA and treated with Scr or E4 peptide (10 μg/mL) for 72 hours. FN, COL1α1, uPAR, and EGR-1 levels were detected in whole cell lysates (upper), and MMP-1, MMP-3, and PAI-1 were detected in culture media supernatants (lower). GAPDH and Ponceau S stain were used as loading controls for lysates and supernatants, respectively. C, control siRNA. (E–G) Plaur–/– mice were treated with PBS (n = 7), BLM (n = 10) (1.5 mU/g), or BLM with E4 (n = 10) (20 μg/mouse). Lung tissues were collected after 14 days. BLM, bleomycin. (E) Collagen content quantified by hydroxyproline assay. (F) mRNA levels of Fn and Col1a1 were measured relative to the housekeeping gene Gapdh. (G) FN, Col1α1, uPA, and PAI-1 protein levels in lung homogenates of Plaur–/– mice detected by immunoblotting. Western blots (left) and graphical presentation of data (right) are shown. β-Actin was used as a loading control. Samples were run in parallel. Statistical analysis was performed using 2-tailed unpaired Student’s t test and 1-way ANOVA as appropriate; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars are mean ± SD.