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.
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Research Article Cell biology Pulmonology

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

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Figure 3

Identification of LOXL2 as the target of EA and corilagin; requirement for active LOXL2 for corilagin-induced inhibition of EMT and Snail expression.

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Identification of LOXL2 as the target of EA and corilagin; requirement f...
(A) LTQ cycle. LTQ converts lysine to allysine and yields an aminophenol intermediate. Subsequent hydrolysis release allysine and the original cofactor, producing hydrogen peroxide and ammonia as side products. (B) Primary human lung fibroblasts cultured in the presence of vitamin C and dextran sulfate were treated with recombinant human LOXL2 and different inhibitors for 7 days. The insoluble cross-linked collagen was extracted and measured by Sircol assay. SB, SB431542, TβRI inhibitor; NAC, N-acetylcysteine, antioxidant. STD, standard. (C) Recombinant human LOXL2 was incubated with 2 mM d-penicillamine (DPA) or different concentrations of corilagin (0–1 μM) for 1 hour, and LOX activity was measured. Data represent mean ± SD; n = 3. (D) NMuMG cells overexpressing human LOXL2 were incubated with or without 0.5 μM corilagin for 24 hours, lysed, and immunoblotted for LOXL2, Snail1, and β-actin. (E) A549 cells transfected with siRNA to LOXL2 were stimulated with TGF-β1 or left unstimulated for 48 hours in the presence or absence of 1 μM corilagin. The lysates were immunoblotted for LOXL2, fibronectin, E-cadherin, Snail1, and β-actin. (F) Primary human lung fibroblasts transfected with siRNAs to LOXL1 or LOXL2 were stimulated with TGF-β1 or left unstimulated for 72 hours in the presence or absence of 1 μM corilagin, and the lysates were immunoblotted for LOXL1, LOXL2, N-cadherin, α-smooth muscle actin (α-SMA), Snail1, and β-actin. B and D–F are representative of at least 3 experiments with similar results.