Ailanthone ameliorates pulmonary fibrosis by suppressing JUN-dependent MEOX1 activation

Abstract

Pulmonary fibrosis poses a significant health threat with very limited therapeutic options available. In this study, we reported the enhanced expression of mesenchymal homobox 1 (MEOX1) in pulmonary fibrosis patients, especially in their fibroblasts and endothelial cells, and confirmed MEOX1 as a central orchestrator in the activation of profibrotic genes. By high-throughput screening, we identified Ailanthone (AIL) from a natural compound library as the first small molecule capable of directly targeting and suppressing MEOX1. AIL demonstrated the ability to inhibit both the activation of fibroblasts and endothelial-to-mesenchymal transition of endothelial cells when challenged by transforming growth factor-β1 (TGF-β1). In an animal model of bleomycin-induced pulmonary fibrosis, AIL effectively mitigated the fibrotic process and restored respiratory functions. Mechanistically, AIL acted as a suppressor of MEOX1 by disrupting the interaction between the transcription factor JUN and the promoter of MEOX1, thereby inhibiting MEOX1 expression and activity. In summary, our findings pinpointed MEOX1 as a cell-specific and clinically translatable target in fibrosis. Moreover, we demonstrated the potent anti-fibrotic effect of AIL in pulmonary fibrosis, specifically through the suppression of JUN-dependent MEOX1 activation.

Keywords: Ailanthone; High-throughput screening; JUN; MEOX1; Natural product; Pulmonary fibrosis; TGF-β1.

Graphical abstract

Figure 1

MEOX1 expression was significantly upregulated during pulmonary fibrosis. (A–B) MEOX1 expression in lung tissues from non-diseased control (NDC) donors, idiopathic pulmonary fibrosis (IPF) patients and interstitial lung disease (ILD) patients were compared by RNA-seq (GSE213001). (C) Representative immunofluorescence images of lung tissue sections from normal and BLM-induced mice showing Meox1 expression, scale bars 100 μm, n = 6. The fluorescence intensity was quantified by ImageJ software. (D) The expressions of Meox1 and fibrotic markers Collagen I and Fibronectin in lung tissues from normal and BLM-induced mice were analyzed by Western blotting and quantified, n = 3. (E) The relative mRNA levels of ACTA2, COL1A1, FN1, CTGF and POSTN in MRC-5 cells infected with lentivirus overexpressing MEOX1 or control vector, n = 6. (F) The relative mRNA levels of MEOX1, ACTA2, COL1A1, and CTGF in MRC-5 cells transfected with siRNA knocking down MEOX1 with or without TGF-β1 induction, n = 3. Data are presented as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 by two-tailed Student's t-test.

Figure 2

Ailanthone was a potent MEOX1 suppressor. (A) Sketch of the gene structure of human MEOX1. Transcriptional start site (TSS) and promoter regions with different length were shown. (B) HeLa cells transfected with reporters containing various length of MEOX1 promoter regions were measured by luciferase activities. (C) Schematic of the MEOX1 reporter construct used in high-throughput screening. (D) Summary of MEOX1 reporter activities evaluating all natural compounds screened in this study against MEOX1 P2 promoter. The ratio between firefly luciferase and renal luciferase was calculated and normalized to DMSO controls. (E) Changes of MEOX1 expression by hit compounds in HeLa cells. The mRNA level of MEOX1 in all groups was normalized to DMSO group. (F) The mRNA level of Meox1 was determined in primary mouse lung fibroblasts (PMLFs) incubated with hit compounds (10 μmol/L) after TGF-β1 (5 ng/mL) stimulation. (G) Chemical structures of AIL. (H) The expression of MEOX1 was measured in the presence of varying concentrations of AIL by qRT-PCR and IC₅₀ was determined, n = 3. (I) The protein level of MEOX1 in HeLa cells treated with varying concentrations of AIL for 24 h was analyzed by Western blotting and quantified, n = 3. Data are presented as mean ± SD; ^##P < 0.01 by two-tailed Student's t-test; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 by one-way ANOVA followed by Tukey's post hoc test between indicated groups.

Figure 3

Ailanthone suppressed proliferation and migration of pulmonary fibroblasts under TGF-β1 stimulation. (A) MRC-5 cells were incubated with various concentrations of AIL for 24 h and then harvested for LDH release assay, n = 6. (B) The concentration-dependent inhibitory effect of AIL on MRC-5 cells under TGF-β1 stimulation was determined by CCK-8, n = 4. (C) Representative images of EdU assay on AIL-treated MRC-5 cells, cell nuclei stained with DAPI are shown in blue, and EdU-positive cell nuclei are shown in red, Scale bars: 100 μm. (D) Statistics of the EDU-positive ratio was analyzed with Thermo Scientific CellInsight CX7 LZR software. (E) Migration of AIL-treated MRC-5 cells were detected by wound healing assay, Scale bars: 100 μm. (F) Statistics of the wound area from (E); n = 6. Data are presented as mean ± SD; ^#P < 0.05, ^###P < 0.001, ^####P < 0.0001 by two-tailed Student's t-test; ∗∗P < 0.01, ∗∗∗∗P < 0.0001 by one-way ANOVA followed by Tukey's post hoc test between indicated groups.

Figure 4

Ailanthone inhibited lung fibroblasts activation and fibrogenesis induced by TGF-β1. (A, B) PMLFs were stimulated with TGF-β1 (5 ng/mL) and treated with AIL (1 μmol/L) for 24 h. The relative mRNA levels of Meox1, Col1a1, Acta2, Fn1 and Ctgf were measured by the qRT-PCR; n = 3. (C, D) PMLFs were stimulated with TGF-β1 (5 ng/mL) and treated with the indicated concentrations of AIL for 24 h. The relative protein levels of Meox1, α-SMA and Fibronectin were analyzed by Western blotting and quantified, n = 3. (E) Representative immunofluorescence images of MRC-5 cells stimulated with TGF-β1 (5 ng/mL) and treated with the indicated concentrations of AIL or JQ-1 for 24 h showing collagen I expression, Scale bars: 100 μm. (F) The relative fluorescence intensity of collagen I was analyzed with ImageJ software; n = 9. (G) Representative immunofluorescence images of MRC-5 cells stimulated with TGF-β1 (5 ng/mL) and treated with the indicated concentrations of AIL or JQ-1 for 24 h showing α-SMA expression, Scale bars: 100 μm. (H) The relative fluorescence intensity of α-SMA was analyzed with ImageJ software; n = 9. (I) The fibroblast-to-myofibroblast conversion ratio in (G) was analyzed with Thermo Scientific CellInsight CX7 LZR software by determining α-SMA-positive cells (myofibroblast) and DAPI-positive cells (total fibroblast). (J) Gene Ontology (GO) analysis of changed genes in MRC-5 cells stimulated with TGF-β1 (5 ng/mL) and treated with or without AIL (1 μmol/L) for 24 h. Log2fold-change > 1, padj < 0.05. (K) Heatmaps of profibrotic marker, cell-cycle, collagen secretion and matrix metalloproteinases (MMPs) related genes in control group, TGF-β1 group (5 ng/mL) and TGF-β1 group with AIL (1 μmol/L) treatment for 24 h. Data are presented as mean ± SD; ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 by two-tailed Student's t-test; ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 by one-way ANOVA followed by Tukey's post hoc test between indicated groups.

Figure 5

Ailanthone suppressed EndMT, barrier permeability and migration of endothelial cells under TGF-β1 stimulation. (A) UMAP representation of MEOX1 expression in endothelial cells isolated from lungs of 20 pulmonary fibrosis patients and 10 normal donors (dataset GSE135893). (B, C) The relative mRNA levels of MEOX1, ACTA2, VIM, CD31 and CDH5 in HPMECs infected with lentivirus overexpressing MEOX1 or control vector, n = 6. (D) The relative mRNA levels of EndMT markers CD31, VIM and ACTA2 in HPMECs treated as indicated were measured by qRT-PCR; n = 3. (E) Immunofluorescence analysis for CD31 (red) and α-SMA (green) in HPMECs treated as indicated, Scale bars: 100 μm. (F) The relative fluorescence intensity of CD31 and α-SMA was analyzed with ImageJ software; n = 9. (G) Schematic of the permeability assay of HPMECs monolayers. (H) Permeability of HPMECs monolayers treated with TGF-β1 (5 ng/mL) and AIL as indicated was quantified based on fluorescence intensity of FITC in the culture media in the lower chambers; n = 6. (I) Wound healing assay was performed on HPMECs following treated with TGF-β1 (5 ng/mL) and AIL as indicated, representative images are shown, scale bars: 100 μm. (J) Quantification of the cell migration rate; n = 6. Data are presented as mean ± SD; ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 by two-tailed Student's t-test; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 by one-way ANOVA followed by Tukey's post hoc test between indicated groups.

Figure 6

AIL attenuates Bleomycine-induced pulmonary fibrosis in mice. (A) Schematic diagram outlining the treatment strategy involving AIL or JQ-1 post bleomycin (BLM)-induced pulmonary fibrosis. (B) Enhanced pause (Penh) was assessed by WBP in each group on Day 21 after modeling; n = 6. (C) Representative images of HE staining of the lung section at Day 21 after BLM-induction, Scale bars: 300 μm. (D) Ashcroft score analysis of (C), n = 6 mice per group, 2 slices per mice. (E) Representative images of Masson's trichrome staining of the lung section on Day 21 after BLM-induction, Scale bars: 300 μm. (F) Collagen volume fraction analysis of (E); n = 6 mice per group, 2 slices per mice. (G) The mRNA levels of EndMT markers Cd31, Cdh5 and Vim in lung tissues on Day 21 after BLM-induction were analyzed by qRT-PCR; n = 3. (H) The mRNA levels of Meox1 and fibrotic markers Acta2, Col1a1 and Fn1 in lung tissues on Day 21 after BLM-induction were analyzed by qRT-PCR; n = 9. (I) The expression of Meox1, Collagen I, Fibronectin and Cd31 in lung tissues of mice was analyzed by Western blotting. (J) Quantitative analysis of protein expression in (I) was performed by ImageJ software; n = 6. (K) Quantitative hydroxyproline assay of the right lung in the groups as indicated; n = 6. Data are presented as mean ± SD; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 by two-tailed Student's t-test; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001 by one-way ANOVA followed by Tukey's post hoc test between indicated groups.

Figure 7

AIL suppressed MEOX1 transcription through JUN. (A) HeLa cells transfected with control vector or JUN-overexpressing plasmid were detected by dual luciferase reporter assays to reflect MEOX1 promoter activity; n = 4. (B) HeLa cells were transfected with siRNA targeting JUN (si-JUN) or a scrambled control (si-NC) for 24 h, mRNA levels of JUN and MEOX1 was analyzed by qRT-PCR; n = 3. (C, D) HeLa cells transfected with control vector or JUN-overexpressing plasmid were treated with indicated concentrations of AIL for 24 h, and then the expression of MEOX1 was analyzed by qRT-PCR (C) and the activity of MEOX1 promoter was determined by dual luciferase reporter assay (D); n = 3. (E) Three potential JUN binding sites (BS) within MEOX1 promoter were predicted by JASPAR. The relative location of BS sites in MEOX1 gene and primers used in ChIP-qPCR to detect these sites were shown. (F) HeLa cells were transfected with P2-Fluc-Rluc (P2) or P2-ΔBS123, P2-ΔBS1, P2-ΔBS2, P2-ΔBS3 in which either one or all three BS sites were removed from P2, 48 h post-transfection dual luciferase reporter assays were performed; n = 4. (G) HeLa cells were transfected with P2-ΔBS2, 24 h post-transfection, cells were treated with indicated concentrations of AIL for 24 h, activity of MEOX1 promoter were detected by dual luciferase reporter assays; n = 4. (H) ChIP-qPCR assays for JUN binding ability to three BS sites in MEOX1 promoter in HeLa cells. (I) ChIP-qPCR assays for JUN binding to the BS2 site (ATTAATCATC) in MEOX1 promoter in HeLa cells with or without AIL treatment. (J) Agarose gel electrophoresis of the qPCR products in (I). (K) Biotin-labeled DNA probes containing BS1, 2 and 3 sequence within MEOX1 promoter or 100-fold molar excess of unlabeled competitors (cold probes) were incubated with HeLa nuclear extracts, EMSA results was used to illustrate the specificity of the protein/DNA complexes. (L) Biotin-labeled DNA probe containing BS2 sequence was incubated with or without HeLa nuclear extracts, in the absence or presence of increasing concentrations of AIL. EMSA results was used to illustrate the effect of AIL on the association of protein and BS2. (M, N) The 2D and 3D residue interactions of the JUN/FOS/BS2 complex with AIL are depicted. The JUN/FOS/BS2 complex is illustrated as red and white ribbons with residues forming interaction shown as dashed lines. AIL is depicted in green. Data are presented as mean ± SD; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, ^####P < 0.0001 by two-tailed Student's t-test; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗∗P < 0.0001 by one-way ANOVA followed by Tukey's post hoc test between indicated groups.

Figure 8

Cartoon depicts how Ailanthone ameliorates pulmonary fibrosis by suppressing JUN-dependent MEOX1 activation. Injury and/or stress induce the recruitment of the AP-1 transcription complex to the MEOX1 gene through binding of JUN to the ATTAATCATC motif on the MEOX1 promoter. This enhances MEOX1 expression, subsequently causing the activation of fibroblasts and EndMT of endothelial cells, ultimately leading to pulmonary fibrosis. Ailanthone functions as a potent MEOX1 suppressor by disrupting the interaction between JUN and the MEOX1 promoter. This disruption inhibits MEOX1 expression and activity, resulting in the amelioration of pulmonary fibrosis both in vitro and in vivo.