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 6

rENO promotes fibrosis in mouse and human lungs.

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rENO promotes fibrosis in mouse and human lungs. VC or rENO (10 μg/dose)...
VC or rENO (10 μg/dose) were administered intratracheally to 6- to 8-week-old C57BL/6J mice (n = 5/group). Mice were given a total of 4 doses over a period of 2 weeks. BALF and lungs were collected on day 14 after treatment. (A) Lung sections of VC- and rENO-treated mice were stained with Masson’s trichrome blue. Scale bars: 50 μm. (B) mRNA expression levels of Fn, Col1a1, and Plau were measured and shown relative to the housekeeping gene Hprt1. (C) Protein levels of FN and COL1α1 in mouse lung homogenates were detected by immunoblotting. Western blots (left) and graphical presentation of the data (right) are shown. GAPDH was used as a loading control. Samples were run in parallel. (D) Protein levels of uPA and PAI-1 in BALF of mice treated as above were detected by immunoblotting. Western blots (left) and graphical presentation of uPA and PAI-1 levels and the uPA/PAI-1 ratio (right) are shown. Ponceau S was used to normalize signals. (E) Normal human lung tissues (n = 5 from 4 independent donors) were treated with VC and rENO protein (4 μg) for 96 hours. Protein levels of FN, COL1α1, MMP-1, MMP-3, PAI-1, and αSMA were detected by immunoblotting. Representative Western blots (left) and graphical presentation of the data (right) are shown. GAPDH was used as a loading control. Statistical analysis was performed using 1-tailed unpaired or paired Student’s t test as appropriate; *P < 0.05, **P < 0.01. Error bars are mean ± SD.