Fibroblast-specific inhibition of TGF-β1 signaling attenuates lung and tumor fibrosis
Ying Wei, … , Bradley J. Backes, Harold A. Chapman
Ying Wei, … , Bradley J. Backes, Harold A. Chapman
Published September 5, 2017
Citation Information: J Clin Invest. 2017;127(10):3675-3688. https://doi.org/10.1172/JCI94624.
View: Text | PDF
Research Article Cell biology Pulmonology Article has an altmetric score of 54

Fibroblast-specific inhibition of TGF-β1 signaling attenuates lung and tumor fibrosis

Abstract

TGF-β1 signaling is a critical driver of collagen accumulation and fibrotic disease but also a vital suppressor of inflammation and epithelial cell proliferation. The nature of this multifunctional cytokine has limited the development of global TGF-β1 signaling inhibitors as therapeutic agents. We conducted phenotypic screens for small molecules that inhibit TGF-β1–induced epithelial-mesenchymal transition without immediate TGF-β1 receptor (TβR) kinase inhibition. We identified trihydroxyphenolic compounds as potent blockers of TGF-β1 responses (IC50 ~50 nM), Snail1 expression, and collagen deposition in vivo in models of pulmonary fibrosis and collagen-dependent lung cancer metastasis. Remarkably, the functional effects of trihydroxyphenolics required the presence of active lysyl oxidase–like 2 (LOXL2), thereby limiting effects to fibroblasts or cancer cells, the major LOXL2 producers. Mechanistic studies revealed that trihydroxyphenolics induce auto-oxidation of a LOXL2/3–specific lysine (K731) in a time-dependent reaction that irreversibly inhibits LOXL2 and converts the trihydrophenolic to a previously undescribed metabolite that directly inhibits TβRI kinase. Combined inhibition of LOXL2 and TβRI activities by trihydrophenolics resulted in potent blockade of pathological collagen accumulation in vivo without the toxicities associated with global inhibitors. These findings elucidate a therapeutic approach to attenuate fibrosis and the disease-promoting effects of tissue stiffness by specifically targeting TβRI kinase in LOXL2-expressing cells.

Authors

Ying Wei, Thomas J. Kim, David H. Peng, Dana Duan, Don L. Gibbons, Mitsuo Yamauchi, Julia R. Jackson, Claude J. Le Saux, Cheresa Calhoun, Jay Peters, Rik Derynck, Bradley J. Backes, Harold A. Chapman

×

Figure 1

EA and corilagin inhibit TGF-β1–dependent EMT and attenuate bleomycin-induced fibrogenesis.

Options: View larger image (or click on image) Download as PowerPoint
EA and corilagin inhibit TGF-β1–dependent EMT and attenuate bleomycin-in...
(A) Structure of EA. (B) Immunofluorescence of TGF-β1–stimulated A549 cells treated with DMSO, SB431542 (SB), or EA. Green, E-cadherin; orange, fibronectin; blue, DAPI. Scale bars: 500 μm. Each assay was performed in triplicate. (C) A549 cells were treated with EA (1 μM) and TGF-β1 for 1.5 hours and lysates immunoblotted for p-Smad2, Smad2, and β-actin. Repeat = 3. (D) EA dosing and treatment in lung fibrosis model. EA and control (ctl) chow were given to mice for 21 days. Osmotic pumps with EA or PBS were implanted on mice for 7 days at day 10 after bleomycin. (E) Hydroxyproline analysis of lung tissues from mice given saline ctl chow (n = 4), saline EA chow (n = 4), bleomycin ctl chow (n = 10), and bleomycin EA chow (n = 10). Data represent mean ± SD. (F) Masson’s trichrome staining of lung sections from ctl or EA pump–treated mice 17 days after bleomycin. Mosaic images (×4) covering whole lung section are shown. (G) Structure of corilagin. (H) A549 cells stimulated with TGF-β1 were treated with corilagin (0–5 μM) for 48 hours and lysates blotted for fibronectin, E-cadherin, Snail1, and β-actin. Repeat = 3. (I) Corilagin dosing and treatment in lung fibrosis model. Vehicle or corilagin was given to mice by daily gavage starting from day 10 after bleomycin for 11 days. (J) Hydroxyproline analysis of lung tissues from mice treated with saline vehicle (n = 7), saline corilagin (n = 7), bleomycin vehicle (n = 9), and bleomycin corilagin (n = 9). Data represent mean ± SD. (K) Whole lung lysates from mice given saline vehicle (n = 4), saline corilagin (n = 4), bleomycin vehicle (n = 5), and bleomycin corilagin (n = 5) were blotted for fibronectin, collagen I, Snail1, β-actin, p-Smad3, and total Smad3. Quantification of bands normalized to β-actin is expressed as mean ± SD. Data in E, J, and K were analyzed by 1-way ANOVA with a Tukey post hoc test.