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.

Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1. EA and corilagin inhibit TGF-β1–dependent EMT and attenuate bleomycin-induced fibrogenesis.

(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.

Figure 2. EA-rich diet attenuates 344SQ lung tumor metastasis and primary tumor collagen cross-linking.

(A) Implantation and treatment in syngeneic lung cancer model. Metastatic 344SQ tumor cells were subcutaneously injected in syngeneic mice at 12 weeks old and treated with ctl or EA chow for 5 weeks. (B) Representative pictures of lung metastasis of 344SQ tumors treated with ctl or EA chow. (C) Quantification of lung metastasis of 344SQ tumors treated with ctl or EA chow (n = 14 mice per group). (D) Total volume of 344SQ tumors treated with ctl or EA chow (n = 14 mice per group). (E) Total tumor weight (grams) of ctl or EA chow–treated 344SQ primary tumors (n = 14 mice per group). (F) Collagen I immunohistochemistry (IHC) of 344SQ primary tumors treated with ctl or EA chow. Scale bars: 50 μm. (G) Ctl or EA chow–treated 344SQ primary tumors were lysed and immunoblotted for fibronectin, collagen I, β-actin, p-Smad3, and total Smad3. Quantification of bands normalized to β-actin was pooled from 10 mice per group. Data represent mean ± SD. Each protein was analyzed by 1-way ANOVA with a Tukey post hoc test. (H) Representative second-harmonic generation (SHG) images from 344SQ primary tumors treated with ctl or EA chow. Scale bars: 50 μm. (I) Quantification of curvature ratio for individual collagen fibers imaged by SHG microscopy of primary 344SQ tumor tissues treated with ctl (n = 102 collagen fibers per sample) or EA chow (n = 141 collagen fibers per sample). Data for C–E and I are expressed as mean ± SD. P value by unpaired 2-tailed t test.

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.

(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.

Figure 4. Corilagin inhibition of TGF-β1 signaling is dependent on LOXL2 activity.

(A) A549 cells pretreated with 1 μM corilagin for 0, 3, or 6 hours were stimulated with different doses of TGF-β1 (0, 0.2, or 1 ng/ml) for 30 minutes, and the cell lysates were blotted for p-Smad2, p-Smad3, Smad2, Smad3, and β-actin. (B) NMuMG cells transfected with human LOXL2 or empty vector were pretreated with 1 μM corilagin or DMSO for 6 hours and then incubated without or with TGF-β1 for 30 minutes. The cell lysates were blotted for LOXL2, p-Smad3, Smad3, and β-actin. (C and D) A549 cells transfected with siRNA to LOXL2 (C) and primary human lung fibroblasts transfected with siRNAs to LOXL1 or LOXL2 (D) were pretreated with 1 μM corilagin or DMSO for 6 hours before incubation without or with TGF-β1 for 30 minutes. The cell lysates were blotted for p-Smad3 and Smad3. The ratio of p-Smad3/Smad3 for each lane is shown. (E) Mouse lung epithelial cells, fibroblasts, and immune cells sorted from mice treated for 14 days with saline (S), bleomycin with vehicle control (BC), or bleomycin with 7 days oral EGCG (100 mg/kg) (BE) were immediately treated with TGF-β1 for 30 minutes and cell lysates blotted for p-Smad3, total Smad3, and β-actin. n = 5 for each group. (F) A549 cells were pretreated with 1 μM corilagin with or without 2 mM DPA for 6 hours before TGF-β1 stimulation for 30 minutes. The cell lysates were blotted for p-Smad3, Smad3, and β-actin. The data from A–D and F are representative of at least 3 experiments with similar results.

Figure 5. Trihydroxyphenolic motif mimics the LTQ, leading to auto-oxidation of LOXL2 K731 and inactivation of LOXL2.

(A) Catalytic domain sequence of LOXL2. Catalytic domain: P548–S751. LTQ sites are highlighted and boldface (K653 and Y689). The 3 point mutation sites are in red (K614N, K731R, K759R). (B) NMuMG cells transfected with WT or mutant human LOXL2 were treated with 1 μM corilagin or DMSO for 6 hours. LOX activity of conditioned media from treated cells was measured. Data are presented as percent activity of no-corilagin control. Mean ± SD, n = 3. (C) NMuMG cells transfected with WT or mutant human LOXL2 were pretreated with 1 μM corilagin or DMSO for 6 hours and then incubated without or with TGF-β1 for 30 minutes. The cell lysates were immunoblotted for LOXL2, p-Smad3, Smad3, and β-actin. (D) NMuMG cells transfected with WT or mutant human LOXL2 were incubated with or without 1 μM corilagin for 24 hours, and lysates were immunoblotted for LOXL2, Snail1, and β-actin. (E) NMuMG cells transfected with WT or mutant human LOXL2 were incubated with 1 μM corilagin for 6 hours and labeled with 2.5 mM biotin hydrazide for 2 hours. The biotin hydrazide–linked carbonylated LOXL2 was pulled down with streptavidin–magnetic beads, and the precipitates and input protein were immunoblotted for LOXL2. C–E are representative of at least 3 experiments with similar results.

Figure 6. Generation of a novel nondiffusible TβRI kinase inhibitor.

(A) Structure of pyrogallol (Pg), 3Abd, and derivatives. (B) A549 cells were stimulated with TGF-β1 for 30 minutes and lysates immunoblotted for p-Smad3, Smad3, and β-actin. Treatment without preincubation: 3Abd (0.5–10 μM); 2Abd or 4Abd (1, 10 μM). (C) A549 cells were stimulated with TGF-β1 for 48 hours and lysates immunoblotted for LOXL2, fibronectin, E-cadherin, Snail1, and β-actin. 3Abd was added at concentraions ranging from 0.5 to 10 μM. (D) Purified ALK5/TβRI catalytic domain kinase assay was performed with 10 doses of 3Abd or Pg starting from 100 μM. Kinase activity was indicated by 33P-ATP signals, and IC50 of 3Abd was calculated as approximately 3 μM. B–D are representative of 3 experiments with similar results. (E) SMAD-binding element (SBE) reporter–transfected NMuMG and A549 cells were seeded into a 96-well plate. Cocultured wells were seeded with 5,000 transfected NMuMG cells and 25,000 nontransfected A549 cells. Cells were pretreated with or without 1 μM corilagin, 1 μM EGCG, 10 μM 3Abd, or 5 μM SB431542 for 6 hours and stimulated with TGF-β1 overnight before lysis for luciferase assay. Data are presented as percent TGF-β1–induced SBE luciferase activity of DMSO control in log scale. Mean ± SD, n = 3. (F) Flag-tagged TβRI and TβRII were immunoprecipitated from A549 cells and in vitro kinase assay performed on beads exposed to lysate pretreated with corilagin or DMSO. The final reaction was eluted and analyzed by immunoblotting for phosphotyrosine and TβRI. The phosphotyrosine bands were quantified using ImageJ and normalized to DMSO control. Data represent mean ± SD. P value by unpaired 2-tailed t test of 6 separate experiments. (G) Schematic overview of mechanism. A trihydroxyphenolic-containing compound engages active LOXL2, initiating auto-oxidation of K731 and creating a key allysine inactivating the enzyme. In the process, a 3Abd-like metabolite is generated that then blocks TβRI kinase. The combined effects block pathological collagen accumulation.