Attenuation of lung fibrosis in mice with a clinically relevant inhibitor of glutathione-S-transferase π
David H. McMillan, … , Vikas Anathy, Yvonne M.W. Janssen-Heininger
David H. McMillan, … , Vikas Anathy, Yvonne M.W. Janssen-Heininger
Published June 2, 2016
Citation Information: JCI Insight. 2016;1(8):e85717. https://doi.org/10.1172/jci.insight.85717.
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Research Article Pulmonology Therapeutics

Attenuation of lung fibrosis in mice with a clinically relevant inhibitor of glutathione-S-transferase π

Abstract

Idiopathic pulmonary fibrosis (IPF) is a debilitating lung disease characterized by excessive collagen production and fibrogenesis. Apoptosis in lung epithelial cells is critical in IPF pathogenesis, as heightened loss of these cells promotes fibroblast activation and remodeling. Changes in glutathione redox status have been reported in IPF patients. S-glutathionylation, the conjugation of glutathione to reactive cysteines, is catalyzed in part by glutathione-S-transferase π (GSTP). To date, no published information exists linking GSTP and IPF to our knowledge. We hypothesized that GSTP mediates lung fibrogenesis in part through FAS S-glutathionylation, a critical event in epithelial cell apoptosis. Our results demonstrate that GSTP immunoreactivity is increased in the lungs of IPF patients, notably within type II epithelial cells. The FAS-GSTP interaction was also increased in IPF lungs. Bleomycin- and AdTGFβ-induced increases in collagen content, α-SMA, FAS S-glutathionylation, and total protein S-glutathionylation were strongly attenuated in Gstp–/– mice. Oropharyngeal administration of the GSTP inhibitor, TLK117, at a time when fibrosis was already apparent, attenuated bleomycin- and AdTGFβ-induced remodeling, α-SMA, caspase activation, FAS S-glutathionylation, and total protein S-glutathionylation. GSTP is an important driver of protein S-glutathionylation and lung fibrosis, and GSTP inhibition via the airways may be a novel therapeutic strategy for the treatment of IPF.

Authors

David H. McMillan, Jos L.J. van der Velden, Karolyn G. Lahue, Xi Qian, Robert W. Schneider, Martina S. Iberg, James D. Nolin, Sarah Abdalla, Dylan T. Casey, Kenneth D. Tew, Danyelle M. Townsend, Colin J. Henderson, C. Roland Wolf, Kelly J. Butnor, Douglas J. Taatjes, Ralph C. Budd, Charles G. Irvin, Albert van der Vliet, Stevenson Flemer, Vikas Anathy, Yvonne M.W. Janssen-Heininger

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Figure 4

TLK117-induced GSTP inhibition attenuates bleomycin-induced collagen content.

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TLK117-induced GSTP inhibition attenuates bleomycin-induced collagen con...
(A) Assessment of caspase-3 activity in wild-type and lpr mouse lung fibroblasts transfected with pcDNA3.1, wild-type Fas, or C294A mutant Fas; pretreated with TLK117 for 2 hours; and treated with FASL for 4 hours. *P < 0.05 relative to respective vehicle/control group, †P < 0.05 relative to respective vehicle/FASL group, ‡P < 0.05 relative to respective wild-type FAS construct group by 1-way ANOVA with a Tukey post-test (n = 3 per group, representative of 2 independent experiments). (B) Assessment of caspase-3 activity in mouse type II alveolar epithelial cells following TLK117 pretreatment for 2 hours and treatment with either FASL or TNF-α with cycloheximide for 4 hours. *P < 0.05 relative to respective vehicle/control group, †P < 0.05 relative to respective vehicle/FASL group by 1-way ANOVA with a Tukey post-test (n = 6 per group, representative of 2 independent experiments). (C) Evaluation of GSTP activity in mouse lungs following oropharyngeal administration of either 25 or 50 mg/kg TLK117 for 4 or 24 hours. *P < 0.05 relative to vehicle control by 1-way ANOVA (n = 3 per group). (D) Schematic of the TLK117 treatment strategy with the bleomycin model. (E and F) Assessment of hydroxyproline and soluble collagen content in mouse lung homogenates. *P < 0.05 relative to PBS group, †P < 0.05 relative to 15-day bleomycin group, ‡P < 0.05 relative to 28-day bleomycin/vehicle group by 1-way ANOVA with a Tukey post-test. Shown are pooled data from 2 independent experiments (n = 6 per group). (G) Assessment of fibrotic remodeling by Masson’s trichrome staining. (H) Assessment of α-SMA immunoreactivity by immunohistochemistry (red, α-SMA, blue, hematoxylin). Scale bar: 100 μm. lpr, Fas-deficient; GSTP, glutathione-S-transferase π; α-SMA, α-smooth muscle actin; Bleo, bleomycin; Veh, vehicle; TLK, TLK117; FASL, FAS ligand; TNF, TNF-α; CHX, cycloheximide; RLU, relative luminescence units; Euth, euthanasia.