The antifibrotic effects of plasminogen activation occur via prostaglandin E2 synthesis in humans and mice
Kristy A. Bauman, Scott H. Wettlaufer, Katsuhide Okunishi, Kevin M. Vannella, Joshua S. Stoolman, Steven K. Huang, Anthony J. Courey, Eric S. White, Cory M. Hogaboam, Richard H. Simon, Galen B. Toews, Thomas H. Sisson, Bethany B. Moore, Marc Peters-Golden
Kristy A. Bauman, Scott H. Wettlaufer, Katsuhide Okunishi, Kevin M. Vannella, Joshua S. Stoolman, Steven K. Huang, Anthony J. Courey, Eric S. White, Cory M. Hogaboam, Richard H. Simon, Galen B. Toews, Thomas H. Sisson, Bethany B. Moore, Marc Peters-Golden
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Research Article Pulmonology

The antifibrotic effects of plasminogen activation occur via prostaglandin E2 synthesis in humans and mice

Abstract

Plasminogen activation to plasmin protects from lung fibrosis, but the mechanism underlying this antifibrotic effect remains unclear. We found that mice lacking plasminogen activation inhibitor–1 (PAI-1), which are protected from bleomycin-induced pulmonary fibrosis, exhibit lung overproduction of the antifibrotic lipid mediator prostaglandin E2 (PGE2). Plasminogen activation upregulated PGE2 synthesis in alveolar epithelial cells, lung fibroblasts, and lung fibrocytes from saline- and bleomycin-treated mice, as well as in normal fetal and adult primary human lung fibroblasts. This response was exaggerated in cells from Pai1–/– mice. Although enhanced PGE2 formation required the generation of plasmin, it was independent of proteinase-activated receptor 1 (PAR-1) and instead reflected proteolytic activation and release of HGF with subsequent induction of COX-2. That the HGF/COX-2/PGE2 axis mediates in vivo protection from fibrosis in Pai1–/– mice was demonstrated by experiments showing that a selective inhibitor of the HGF receptor c-Met increased lung collagen to WT levels while reducing COX-2 protein and PGE2 levels. Of clinical interest, fibroblasts from patients with idiopathic pulmonary fibrosis were found to be defective in their ability to induce COX-2 and, therefore, unable to upregulate PGE2 synthesis in response to plasmin or HGF. These studies demonstrate crosstalk between plasminogen activation and PGE2 generation in the lung and provide a mechanism for the well-known antifibrotic actions of the fibrinolytic pathway.

Authors

Kristy A. Bauman, Scott H. Wettlaufer, Katsuhide Okunishi, Kevin M. Vannella, Joshua S. Stoolman, Steven K. Huang, Anthony J. Courey, Eric S. White, Cory M. Hogaboam, Richard H. Simon, Galen B. Toews, Thomas H. Sisson, Bethany B. Moore, Marc Peters-Golden

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Figure 7

IMR-90 cell PGE2 synthesis is regulated by plasminogen activation.

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IMR-90 cell PGE2 synthesis is regulated by plasminogen activation. (...
(A) IMR-90 cells in SFM were treated with PLG (200 mU/ml), plasmin (100 mU/ml), or uPA (10 U/ml) for 18 hours. Medium was removed from the cells, and PGE2 was measured by ELISA and is expressed relative to the control value measured in SFM alone; n ≥ 5, *P < 0.05 versus control. (B) Cell supernatants were collected from either saline-treated murine lung fibroblasts or human IMR-90 cells cultured at equal densities for 24 hours. Supernatants were then analyzed by zymography for the ability to activate PLG. A band of the appropriate size for human uPA (54 kDa) is readily distinguishable in the IMR-90 cell supernatants, but the lower-molecular-weight murine uPA band (45 kDa) is very faint in the murine lung fibroblasts; n = 5 per group. (C) IMR-90 cells in SFM were treated with 100 mU/ml plasmin for 18 hours. Media was harvested for PGE2 determination (triangles), and lysates were harvested for collagen I protein expression (circles) or COX-2 protein expression (squares) as determined by immunoblot analysis and densitometry using α-tubulin as a loading control; n = 3, *P < 0.05 versus control. Collagen I and COX-2 immunoblots are representative of experiments performed in triplicate. (D) IMR-90 cells were treated with SFM (control), PLG (100 mU/ml), or PLG plus indomethacin (Indo; 10 μM) for 18 hours, and collagen I was determined by immunoblot analysis and densitometry using α-tubulin as a loading control; n = 3, *P < 0.05 versus control. Collagen I immunoblots are representative of experiments performed in triplicate. Blots in both C and D are composed from lanes that were run on the same gel but were noncontiguous.