Research ArticlePULMONARY DISEASES

The FOXM1 inhibitor RCM-1 suppresses goblet cell metaplasia and prevents IL-13 and STAT6 signaling in allergen-exposed mice

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Sci. Signal.  18 Apr 2017:
Vol. 10, Issue 475, eaai8583
DOI: 10.1126/scisignal.aai8583
  • Fig. 1 Identification of the FOXM1 inhibitor RCM-1 by high-throughput screening.

    (A) Schematic of the high-throughput screen. U2OS C3 cells expressing a Dox-inducible GFP-FOXM1 construct were treated with Dox and, 24 hours later, screened with 50,000 small-molecule compounds. AU, arbitrary units. (B) Chemical structure of the RCM-1 compound. (C and D) RCM-1 decreased nuclear GFP-FOXM1 fluorescence. U2OS C3 cells were fixed and imaged for GFP (green) (C). Twelve random images were taken of each cell culture. DNA stain (red) and phase contrast were used to identify the nuclear-cytoplasm boundary in individual cells. Nuclear (nuc)/cytoplasmic (cyto) GFP ratio in U2OS C3 cells is shown as means ± SD (n = 200 cells per condition) (D). Scale bars, 10 μm. (E) Different concentrations of the RCM-1 compound were used to determine EC50 (n = 3 independent cell cultures). GFP-nuc, total GFP fluorescence in cell nuclei; GFP-cyto, total GFP fluorescence in cytoplasm. (F) Decreased mRNA abundance of FOXM1 target genes Cdc25B and Plk1 was found in RCM-1–treated cells by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (n = 3 independent experiments). **P < 0.01. (G) Western blots show amounts of endogenous FOXM1 protein and other transcription factors in RCM-1–treated human airway epithelial cells cultured on air-liquid interface (n = 3 independent experiments). (H) RCM-1 increases ubiquitination of FOXM1 (arrows). FOXM1 immunoprecipitates (IP) from A549 cell lysates were Western-blotted with ubiquitin (Ub) or FOXM1 antibodies. Asterisk indicates the location of immunoglobulin G (IgG) on ubiquitin Western blot. Amounts of ubiquitin-FOXM1 were normalized to total FOXM1 (bottom) (n = 3 independent experiments). (I and J) RCM-1 induces the translocation of endogenous FOXM1 from cell nuclei to cytoplasm. FOXM1 (red) colocalizes with ubiquitin (I) and PSMA5 (J) (green) in proteasomes of RCM-1–treated A549 cells (yellow). 4′,6-Diamidino-2-phenylindole (DAPI) was used to visualize cell nuclei (n = 3 independent experiments). Scale bars, 20 μm.

  • Fig. 2 RCM-1 inhibits FOXM1 in vivo.

    (A) RCM-1 inhibits FOXM1 in the mouse lung. BALB/c mice were treated with either RCM-1 or vehicle. Total lung protein was analyzed by Western blot for endogenous FOXM1 and β-actin. Each lane represents a different mouse. (B) GFP-FOXM1 transgenic mice were given Dox to activate the transgene and were treated with either RCM-1 or vehicle for 2 weeks. Frozen lung sections were analyzed for GFP fluorescence. RCM-1 reduces nuclear GFP-FOXM1 in bronchiolar epithelium. Scale bars, 10 μm (images are representative of five mice per treatment). (C) RCM-1 inhibits GFP-FOXM1 after HDM challenge. Tissue samples were prepared from lungs of Dox-treated GFP-FOXM1 transgenic mice after HDM challenge and RCM-1 treatment. Dox-treated CCSP-rtTA mice were used as controls [wild type (WT)]. Paraffin sections were stained with FOXM1 antibodies (dark brown) and counterstained with nuclear fast red (red). Scale bars, 50 μm (images are representative of four mice per treatment). (D) Western blot shows that RCM-1 decreases GFP and FOXM1 protein abundance in lungs of HDM-treated GFP-FOXM1 mice. Each lane represents a different mouse. (E) RCM-1 decreases GFP fluorescence in GFP-FOXM1 mice after HDM treatment. Frozen lung sections were stained with DAPI. Br, bronchiole. Scale bars, 50 μm (left) and 5 μm (right) (images are representative of four mice per treatment).

  • Fig. 3 RCM-1 inhibits HDM-induced airway hyperreactivity.

    (A) Experimental protocol showing the treatment of BALB/c mice with HDM and RCM-1. (B) RCM-1 ameliorated airway hyperreactivity induced by HDM. BALB/c mice were treated with either RCM-1 or vehicle. Airway mechanics were measured with the flexiVent system. RCM-1 treatment decreased HDM-mediated increase in airway resistance, elastance, and tissue damping. RCM-1 increased pulmonary compliance but did not change Newtonian resistance in HDM-treated lungs (n = 5 mice per treatment). *P < 0.05; **P < 0.01.

  • Fig. 4 RCM-1 prevents goblet cell metaplasia in HDM-treated mice.

    (A) H&E and Alcian blue stainings show reduced numbers of goblet cells in mice treated with RCM-1. Scale bars, 50 μm (images are representative of three mice per treatment). (B) Immunostaining shows decreased abundance of SPDEF, MUC5AC, and FOXM1 after RCM-1 treatment of HDM-challenged mice. Scale bars, 50 μm (images are representative of three mice per treatment). (C) qRT-PCR of total lung RNA. HDM treatment increased expression of Foxm1, Spdef, Foxa3, Agr2, and Muc5ac and decreased expression of Foxa2. RCM-1 reversed the effects of HDM on the mRNA expression of these genes. Data are means ± SEM (n = 3 mice per group). *P < 0.05; **P < 0.01.

  • Fig. 5 Effects of RCM-1 on HDM-mediated lung inflammation.

    (A) RCM-1 did not affect the number of inflammatory cells in BALF. Cells were counted in BALF obtained from mice treated with HDM and RCM-1 (n = 6 mice per group). (B) BALF cells were immunostained for cell surface markers and analyzed using flow cytometry (n = 3 mice per group). (C) Histological assessment of lung tissue. Area of inflammation was measured using lung sections stained with H&E (n = 3 mice per group). (D to F) qRT-PCR was performed on total lung RNA to examine mRNAs encoding chemokines and cytokines, including those mediating dendritic cell activation (IL-12p35 and IL-33), TH2 cytokines (IL-4, IL-5, and IL-13), eosinophilic chemoattractants (Ccr3, Ccl11, and Ccl24), macrophage chemoattractants (Ccr2, Ccl2, Cx3cl1, and Cx3cl1), and bronchoconstrictors (Acta2, Ptgs2, and Ltc4s). mRNA expression was normalized to β-actin mRNA (n = 3 mice per group). N.D., not detected. (G) The Luminex Multiplex xMAP bead-based antibody assay was used to measure the concentrations of IFNγ, IL-4, IL-5, and IL-13 in BALF (n = 6 mice per group). ELISA, enzyme-linked immunosorbent assay. Data are means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

  • Fig. 6 RCM-1 inhibits IL-13–induced goblet cell metaplasia.

    (A and B) RCM-1 decreased goblet cell metaplasia after administration of IL-13. Lung paraffin sections were stained with H&E and Alcian blue (A) or used for immunostaining for FOXM1, SPDEF, and MUC5AC (B). Scale bars, 50 μm (images are representative of four mice per treatment). (C) The flexiVent system was used to measure airway mechanics. RCM-1 inhibited IL-13–mediated airway hyperreactivity and increased lung compliance in IL-13–treated mice (n = 5 mice per group). (D) RCM-1 did not change the number of inflammatory cells in BALF of IL-13–treated mice (n = 5 mice per group). Mac, macrophages; Eos, eosinophils; Neu, neutrophils; Lym, lymphocytes. (E) RCM-1 inhibits expression of genes associated with goblet cell metaplasia. qRT-PCR was performed on total lung RNA to measure expression of Foxm1, Spdef, Foxa3, Foxa2, Scgb1a1, Agr2, and Muc5ac (n = 4 mice per group). Data are means ± SEM. *P < 0.05; **P < 0.01.

  • Fig. 7 RCM-1 inhibits ERK1/2 but does not change the phosphorylation of STAT6.

    (A and B) qRT-PCR shows altered expression of genes in RCM-1–treated lungs. qRT-PCR was performed on total lung RNA (n = 4 mice per group). (C) The Luminex Multiplex assay was used to measure the concentrations of IFNγ, IL-4, IL-5, and IL-33 in BALF (n = 5 mice per group). Data are means ± SEM. *P < 0.05; **P < 0.01. (D to F) RCM-1 decreases the abundance of FOXM1 and ERK1/2 and the phosphorylation (p) of ERK1/2 but does not change IL-13–induced phosphorylation of STAT6. Western blot was performed using total protein extract from mouse lung tissue (n = 3 mice per group) (D), human airway epithelial cells (n = 3 independent experiments) (E), and A549 cells (n = 5 independent experiments) (F).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/475/eaai8583/DC1

    Fig. S1. RCM-1 decreases FOXM1 abundance in U2OS cells and pulmonary airways.

    Fig. S2. Lack of toxicity after RCM-1 treatment.

    Fig. S3. RCM-1 protects bronchiolar epithelial cells from HDM-mediated effects.

    Fig. S4. Identification of inflammatory cells in BALF of HDM-treated mice.

    Fig. S5. RCM-1 protects bronchiolar epithelial cells from IL-13–mediated decreases in the abundance of FOXA2.

    Fig. S6. Lung mechanics in mice treated with IL-13 and RCM-1.

    Fig. S7. Lack of toxicity of RCM-1 in IL-13–treated mice.

    Fig. S8. RCM-1 inhibits nuclear accumulation of FOXM1 protein in A549 cells.

    Fig. S9. U0126 decreases lung inflammation but does not affect goblet cell metaplasia or the abundance of FOXM1.

    Table S1. TaqMan assays for qRT-PCR.

    Table S2. Histological evaluation of inflammatory responses in lung tissue.

    Table S3. Histology scores of lung tissue from HDM-treated mice.

  • Supplementary Materials for:

    The FOXM1 inhibitor RCM-1 suppresses goblet cell metaplasia and prevents IL-13 and STAT6 signaling in allergen-exposed mice

    Lifeng Sun, Xiaomeng Ren, I-Ching Wang, Arun Pradhan, Yufang Zhang, Hannah M. Flood, Bo Han, Jeffrey A. Whitsett, Tanya V. Kalin, Vladimir V. Kalinichenko*

    *Corresponding author. Email: vladimir.kalinichenko{at}cchmc.org

    This PDF file includes:

    • Fig. S1. RCM-1 decreases FOXM1 abundance in U2OS cells and pulmonary airways.
    • Fig. S2. Lack of toxicity after RCM-1 treatment.
    • Fig. S3. RCM-1 protects bronchiolar epithelial cells from HDM-mediated effects.
    • Fig. S4. Identification of inflammatory cells in BALF of HDM-treated mice.
    • Fig. S5. RCM-1 protects bronchiolar epithelial cells from IL-13–mediated decreases in the abundance of FOXA2.
    • Fig. S6. Lung mechanics in mice treated with IL-13 and RCM-1.
    • Fig. S7. Lack of toxicity of RCM-1 in IL-13–treated mice.
    • Fig. S8. RCM-1 inhibits nuclear accumulation of FOXM1 protein in A549 cells.
    • Fig. S9. U0126 decreases lung inflammation but does not affect goblet cell metaplasia or the abundance of FOXM1.
    • Table S1. TaqMan assays for qRT-PCR.
    • Table S2. Histological evaluation of inflammatory responses in lung tissue.
    • Table S3. Histology scores of lung tissue from HDM-treated mice.

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    Citation: L. Sun, X. Ren, I.-C. Wang, A. Pradhan, Y. Zhang, H. M. Flood, B. Han, J. A. Whitsett, T. V. Kalin, V. V. Kalinichenko, The FOXM1 inhibitor RCM-1 suppresses goblet cell metaplasia and prevents IL-13 and STAT6 signaling in allergen-exposed mice. Sci. Signal. 10, eaai8583 (2017).

    © 2017 American Association for the Advancement of Science

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