Inhibition of epithelial-to-mesenchymal transition and pulmonary fibrosis by methacycline

Abstract

A high-throughput small-molecule screen was conducted to identify inhibitors of epithelial-mesenchymal transition (EMT) that could be used as tool compounds to test the importance of EMT signaling in vivo during fibrogenesis. Transforming growth factor (TGF)-β1-induced fibronectin expression and E-cadherin repression in A549 cells were used as 48-hour endpoints in a cell-based imaging screen. Compounds that directly blocked Smad2/3 phosphorylation were excluded. From 2,100 bioactive compounds, methacycline was identified as an inhibitor of A549 EMT with the half maximal inhibitory concentration (IC50) of roughly 5 μM. In vitro, methacycline inhibited TGF-β1-induced α-smooth muscle actin, Snail1, and collagen I of primary alveolar epithelial cells . Methacycline inhibited TGF-β1-induced non-Smad pathways, including c-Jun N-terminal kinase, p38, and Akt activation, but not Smad or β-catenin transcriptional activity. Methacycline had no effect on baseline c-Jun N-terminal kinase, p38, or Akt activities or lung fibroblast responses to TGF-β1. In vivo, 100 mg/kg intraperitoneal methacycline delivered daily beginning 10 days after intratracheal bleomycin improved survival at Day 17 (P < 0.01). Bleomycin-induced canonical EMT markers, Snail1, Twist1, collagen I, as well as fibronectin protein and mRNA, were attenuated by methacycline (Day 17). Methacycline did not attenuate inflammatory cell accumulation or alter TGF-β1-responsive genes in alveolar macrophages. These studies identify a novel inhibitor of EMT as a potent suppressor of fibrogenesis, further supporting the concept that EMT signaling is important to lung fibrosis. The findings also provide support for testing the impact of methacycline or doxycycline, an active analog, on progression of human pulmonary fibrosis.

Figure 1.

High-throughput screen of small-molecule library for epithelial–mesenchymal transition (EMT) inhibitors. (A) Immunofluorescence imaging of A549 cells with and without transforming growth factor (TGF)-β1 (4 ng/ml) for 48 hours in the presence of dimethyl sulfoxide (DMSO), methacycline (Met; 10 μM), or SB431542 (SB; 5 μM). E-cadherin, green; fibronectin, red. Scale bar, 50 μm. (B) Scatter plot showing primary screen of EMT inhibitors. Vehicle controls (DMSO only), green; test compounds in DMSO, red; SB in DMSO, blue. Triangles indicate active compounds (ACTIVE CPD) that inhibit over 50% of E-cadherin loss. (C) A549 cells serum starved overnight were stimulated with TGF-β1 (4 ng/ml) in the presence of compound 1, 2, 3, and 4 (Met; 10 μM) for 1.5 hours and the lysates were blotted for phosphorylated Smad (p-Smad) -2, and p-Smad3, total Smad2, and Smad3 are used as loading control. (D) A549 cells stimulated with TGF-β1 (4 ng/ml) were treated with Met (10 μM) or SB (5 μM) for 48 hours and the lysates were blotted for fibronectin, E-cadherin, and Snail1. β-actin is used as loading control. (E) A549 cells serum starved overnight were stimulated with TGF-β1 (4 ng/ml) in the presence of Met (0–20 μM) for 1.5 hours and the lysates were blotted for p-Smad2 and p-Smad3. Total Smad2 and Smad3 are used as loading control. (D–E) A representative blot of three independent experiments is shown.

Figure 2.

Met attenuates TGF-β1–dependent EMT in primary alveolar epithelial cells (AECs). Primary AECs were plated on fibronectin (Fn) for 5 days to allow latent TGF-β1 activation and the cells to undergo EMT. (A) Primary AECs on Fn were cultured in the presence of Met (5,10 μM) or SB (5 μM) and the lysates were blotted for collagen I, α-smooth muscle actin (α-SMA), Snail1, p-Smad2, total Smad2, p-Smad3, total Smad3, and β-actin. The relative intensity normalized to β-actin is shown. (B) Primary AECs on Fn incubated with Met (10 μM) or vehicle control DMSO were immunostained for E-cadherin (green) and α-SMA (red). Contaminating fibroblasts are indicated with arrows. (C) Primary human AECs on Fn were treated with DMSO, Met (10 μM), or SB (5 μM), and the mRNAs were assessed by quantitative RT-PCR (qRT-PCR) analysis. Collagen I and Snail1 mRNA levels are normalized to β-actin mRNA level. Data are expressed as relative fold change over DMSO (set at 1). Mean ± SEM of three independent experiments is shown, and Student’s t test was performed (*P < 0.05, **P < 0.01, ***P < 0.001). (D) Primary fibroblasts were stimulated with TGF-β1 (4 ng/ml) and cultured in the presence of Met (10, 20 μM) or SB (5 μM) for 48 hours, and the lysates were blotted for Fn, collagen I, α-SMA, Snail1, and β-actin. (A and D) A representative blot of three independent experiments is shown.

Figure 3.

Met blocks TGF-β1–induced c-Jun N-terminal kinase (JNK), p38, and Akt activation, but not Smad-binding element (SBE) or TCF/β-catenin reporter activity. A549 cells expressing SBE-luciferase (luc) (A), TOPFlash, or FOPFlash (B) were serum starved overnight and then treated without or with TGF-β1 (4 ng/ml) in combination with DMSO, SB (5 μM), or Met (10 μM), and luc activity was determined after 24 hours. Relative activity was normalized to Renilla luc and relative fold change over DMSO without TGF-β1 (set at 1) was calculated. (A and B) Mean ± SEM of three independent experiments is shown, and Student’s t test was performed. (**P < 0.01, ***P < 0.001; ns, not significant). (C) A549 cells serum-starved for 2 days were treated without or with TGF-β1 (4 ng/ml) in combination with DMSO (−), SB (5 μM), or Met (10 μM) for 15 minutes. Cell lysates were analyzed by immunoblotting with antibodies to p-JNK, JNK, p-p38, p38, extracellular signal–regulated kinase (ERK), p-ERK, p-Akt, and Akt. (D) A549 cells were treated without or with TGF-β1 (4 ng/ml) in combination with DMSO (−), Met (10 μM), JNK inhibitor (10 μM), p38 inhibitor (10 μM), or ERK inhibitor (10 μM) for 48 hours. Cell lysates were immunoblotted for Fn, Snail1, N-cadherin, vimentin, and β-actin. (E) Primary AECs on Fn were cultured in the presence of Met, JNK inhibitor, p38 inhibitor, or ERK inhibitor at 10 μM final concentration, and the lysates were blotted for α-SMA, Snail1, and β-tubulin. (C–E) A representative blot of three independent experiments is shown.

Figure 4.

Met attenuates TGF-β1–stimulated EMT and bleomycin (Bleo)-induced fibrogenesis. (A) Mice were injected intratracheally with Bleo (2.5 U/kg). After 10 days, Met 100 mg/kg or vehicle alone was administered intraperitoneally daily for 7 days. The survival of Bleo-treated mice by Day 17 was plotted and analyzed. (B) EMT markers measured in protein extracts from snap-frozen mouse lungs treated with saline, Bleo, or Bleo + Met. Tissue lysates were blotted for collagen I, Fn, Twist1, Snail1, and β-actin (upper panel). Densitometry values of collagen I, Fn, Twist1, and Snail1 blots were normalized to that of β-actin, and the normalized values of saline were set at 1. Quantification (mean ± SEM) of EMT markers from three independent experiments (total of 8 mice given saline, 15 given Bleo, and 17 given Bleo + Met) is shown in the lower panel. (C) Total lung collagen levels measured by Sircol assay. Pepsin- and acid-soluble collagen content from mice treated with saline + Met, Bleo, and Bleo + Met was normalized to that of saline-treated lungs. Mean ± SEM of the fold changes from three independent experiments was quantified. (D) Collagen I level measured in bronchoalveolar lavage (BAL) fluid by immunoblots. BAL supernatant (50 μl) from mice treated with saline, Bleo, and Bleo + Met was blotted for collagen I (upper panel). Quantification (mean ± SEM) of the densitometry values of collagen I from three independent experiments is shown in the lower panel. (E) Low-power section of 17-day Bleo-injured lung without or with 7-day Met immunostained with E-cadherin (green) and α-SMA (red). Saline-treated lung section was used as staining control. Scale bar, 200 μm. (B and D) A representative blot of three independent experiments is shown. (B–D) *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant by t test.

Figure 5.

Fibrogenic response in mouse BAL fluid. (A) Total protein concentration (μg/lung) from BAL 17 days after intratracheal saline (n = 7), Bleo (n = 11), or Bleo + Met (n = 7). Cell counts (B) and immune cell distribution (C) from BAL of mice intratracheally injected with saline (n = 4), Bleo (n = 9), or Bleo + Met (n = 5). (D) Quantification (mean ± SEM) of p-Smad2 protein from three independent experiments (total of 8 mice given saline, 10 given Bleo, and 15 given Bleo + Met) analyzed by immunoblotting BAL cell pellets. Data are expressed as relative fold change over saline values (set at 1). (E) Transcripts of TGF-β1–responsive genes were measured in RNA extracts from BAL pellets treated with saline (n = 3), Bleo (n = 5), or Bleo + Met (n = 5) by qRT-PCR analysis. Osteopontin (OPN), metalloproteinase (MMP)-19, plasminogen activator type I (PAI)-1, and TREM1 mRNA levels are normalized to β-actin mRNA level. (A–E) *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant by t test.

Figure 6.

Doxyclycline (Dox) inhibits TGF-β1–induced EMT in A549 and lung epithelial cells. (A) Two-dimensional structures of Met, Dox, and tetracycline (Tet). (B) A549 cells were treated without or with TGF-β1 (4 ng/ml) in combination with DMSO (−), Met (10 μM), Dox (10 μM), Tet (10 μM), or SB (5 μM) for 48 hours. Cell lysates were immunoblotted for Fn, E-cadherin, Snail1, N-cadherin, vimentin, and β-actin. (C) Primary AECs on Fn were cultured in the presence of GM6001 (GM) (10 μM), SB (5 μM), DMSO (−), Met (10 μM), Dox (10 μM), or Tet (10 μM), and the lysates were blotted for α-SMA, Snail1, and β-tubulin. (D) Primary AECs on Fn incubated with DMSO, GM (10 μM), Dox (10 μM), or Tet (10 μM) were immunostained for E-cadherin (green) and α-SMA (red). Scale bar, 200 μm. (E) A549 cells were treated without or with TGF-β1 (4 ng/ml) in combination with DMSO (−), GM (10 μM), SB (5 μM), or Met (10 μM) for 48 hours. Cell lysates were immunoblotted for Fn, E-cadherin, Snail1, N-cadherin, vimentin, and β-actin. (B, C, and E) A representative blot of three independent experiments is shown. The relative intensity normalized to β-actin is labeled.