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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

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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 3

E4 increases MMP-1 and reduces COL1α1, FN, and PAI-1 ex vivo.

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E4 increases MMP-1 and reduces COL1α1, FN, and PAI-1 ex vivo.
(A) Ex viv...
(A) Ex vivo model using human lung tissue cores in organ culture. (B) Normal human lung tissues (n = 3/group) were treated with TGF-β1 (10 ng/mL) in combination with Scr or E4 peptide (10 μg/mL) for 120–144 hours. Protein levels of FN, COL1α1, MMP-1, and PAI-1 were analyzed by immunoblotting of whole tissue homogenates. Representative Western blots (left) and quantitative analysis (right). (C and D) SSc (n = 3) and IPF (n = 6) lung tissue cores were treated with Scr or E4 peptide for 96 hours. The amount of collagen in the lungs was quantified using hydroxyproline assay. (E) Lung tissues from patients with SSc (n = 3) were treated with Scr or E4 peptide (10 μg/mL) for 72 hours. Representative Western blots (left) and quantitative analysis (right). GAPDH was used as a loading control. Statistical analysis was performed using 1-tailed unpaired Student’s t test and 1-way ANOVA as appropriate; *P < 0.05, **P < 0.01, ****P < 0.0001. Error bars are mean ± SD.

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