PAI-1 interaction with sortilin-related receptor 1 is required for lung fibrosis
Thomas H. Sisson, John J. Osterholzer, Lisa Leung, Venkatesha Basrur, Alexey Nesvizhskii, Natalya Subbotina, Mark Warnock, Daniel Torrente, Ammara Q. Virk, Sergey S. Gutor, Jeffrey C. Horowitz, Mary Migliorini, Dudley K. Strickland, Kevin K. Kim, Steven K. Huang, Daniel A. Lawrence
Thomas H. Sisson, John J. Osterholzer, Lisa Leung, Venkatesha Basrur, Alexey Nesvizhskii, Natalya Subbotina, Mark Warnock, Daniel Torrente, Ammara Q. Virk, Sergey S. Gutor, Jeffrey C. Horowitz, Mary Migliorini, Dudley K. Strickland, Kevin K. Kim, Steven K. Huang, Daniel A. Lawrence
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Research Article Aging Pulmonology

PAI-1 interaction with sortilin-related receptor 1 is required for lung fibrosis

Abstract

Mutation studies of plasminogen activator inhibitor 1 (PAI-1) have previously implied that PAI-1 promotes lung fibrosis via a vitronectin-dependent (VTN-dependent) mechanism. In the present study, employing 2 distinct murine fibrosis models and VTN-deficient mice, we found that VTN is not required for PAI-1 to drive lung scarring. This result suggested the existence of a profibrotic interaction involving the VTN-binding site on PAI-1 with an unidentified ligand. Using an unbiased proteomic approach, we identified sortilin-related receptor 1 (SorLA) as the most highly enriched PAI-1 binding partner in the fibrosing lung. Investigating the role of SorLA in pulmonary fibrosis demonstrated that deficiency of this protein protected against lung scarring in a murine model. We further found that SorLA is required for PAI-1 to promote scarring in mice, that both SorLA and PAI-1 protein levels are increased in human idiopathic pulmonary fibrosis (IPF) explants, and that these proteins are associated in IPF tissue. Finally, confocal microscopy showed that expression of SorLA in CHO cells increased cellular uptake of PAI-1, and these proteins colocalized in the cytoplasm. Together, these data elucidate a mechanism by which the potent profibrotic mediator PAI-1 drives lung fibrosis and implicate SorLA as a potential therapeutic target in IPF treatment.

Authors

Thomas H. Sisson, John J. Osterholzer, Lisa Leung, Venkatesha Basrur, Alexey Nesvizhskii, Natalya Subbotina, Mark Warnock, Daniel Torrente, Ammara Q. Virk, Sergey S. Gutor, Jeffrey C. Horowitz, Mary Migliorini, Dudley K. Strickland, Kevin K. Kim, Steven K. Huang, Daniel A. Lawrence

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Figure 4

PAI-1WT binds to SorLA in lung tissue homogenates from patients with IPF.

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PAI-1WT binds to SorLA in lung tissue homogenates from patients with IPF...
(A) Fibrotic lung tissue obtained from explants at the time of transplant were homogenized in binding buffer. Each sample (200 μg) was incubated with either uncoated magnetic streptavidin-Sepharose beads or beads coated with biotin-tagged PAI-1WT. Beads were collected, washed, and proteins were eluted with SDS loading buffer. The initial homogenate (input) and the eluted proteins (Beads, PAI-1-Beads) were separated by SDS-PAGE, blotted, and stained with an anti-SorLA antibody. Data are displayed as a representative gel. (B) Equal quantities of protein from homogenized IPF or normal control lung tissue were separated by SDS-PAGE and analyzed by Western blotting for SorLA and αSMA (normalized to vinculin, n = 13). (C) Quantification of SorLA and αSMA in fibrotic tissue. (D) Active PAI-1 levels measured by ELISA (normalized to total lung protein concentration, n = 13). (E–K) Immunofluorescent costaining of PAI-1 (Akoya Opal 520, green) and SorLA (Akoya Opal 690, red) in normal (E and F) and IPF (G–K) human lung tissue sections. (I–K) Enlargement of panel H with (I) DAPI and PAI-1, (J) DAPI and SorLA, and (K) merged. Scale bars: 20 μm. Data are represented as mean ± SEM. Significant P values are shown for comparisons performed using a parametric 2-tailed t test (Panel C, D) or Mann–Whitney U nonparametric 2-tailed t test.