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Proliferation Genes Repressed by TGF-β Are Downstream of Slug/Snail2 in Normal Bronchial Epithelial Progenitors and Are Deregulated in COPD

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Abstract

Slug/Snail2 belongs to the Epithelial-Mesenchymal Transition (EMT)-inducing transcription factors involved in development and diseases. Slug is expressed in adult stem/progenitor cells of several epithelia, making it unique among these transcription factors. To investigate Slug role in human bronchial epithelium progenitors, we studied primary bronchial basal/progenitor cells in an air-liquid interface culture system that allows regenerating a bronchial epithelium. To identify Slug downstream genes we knocked down Slug in basal/progenitor cells from normal subjects and subjects with COPD, a respiratory disease presenting anomalies in the bronchial epithelium and high levels of TGF-β in the lungs. We show that normal and COPD bronchial basal/progenitors, even when treated with TGF-β, express both epithelial and mesenchymal markers, and that the epithelial marker E-cadherin is not a target of Slug and, moreover, positively correlates with Slug. We reveal that Slug downstream genes responding to both differentiation and TGF-β are different in normal and COPD progenitors, with in particular a set of proliferation-related genes that are among the genes repressed downstream of Slug in normal but not COPD. In COPD progenitors at the onset of differentiation in presence of TGF-β,we show that there is positive correlations between the effect of differentiation and TGF-β on proliferation-related genes and on Slug protein, and that their expression levels are higher than in normal cells. As well, the expression of Smad3 and β-Catenin, two molecules from TGF-βsignaling pathways, are higher in COPD progenitors, and our results indicate that proliferation-related genes and Slug protein are increased by different TGF-β-induced mechanisms.

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Data Availability

The data analyzed in this publication have been deposited in the NCBI’s Gene.

Expression Omnibus (GEO) and will be accessible through GEO Series Accession.

Number: GSE122957 and GSE123129.

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Acknowledgements

We thank the Pulmonary Department, the Pathology Department and the Thoracic Surgery Department at Bichat-Claude Bernard University Hospital (Paris, France) and INSERM UMR 1152 for providing lung tissues and isolating the cells. We thank Audrey Rapinat and David Gentien at the Genomics platform, Curie Institute (Paris, France) for Affymetrix GeneChip hybridization. We thank Dusko Ilic and Pierre Savagner for critical reading of the manuscript and helpful discussions.

Funding

PL is supported by the French National Center for Scientific Research (CNRS). This work was supported by a donation from Association Science et Technologie (Groupe Servier) to PL and by funding from French National Institute for Medical Research (INSERM).

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Authors and Affiliations

  1. INSERM UMR1152, Physiopathology and Epidemiology of Respiratory Diseases, Paris, France

    Chamseddine Ben Brahim, Charlotte Courageux & Pascale Leroy

  2. Faculty of Medicine, Paris Diderot University, Bichat Campus, Paris, France

    Chamseddine Ben Brahim, Charlotte Courageux & Pascale Leroy

  3. GenoSplice, Paris, France

    Ariane Jolly & Pierre de la Grange

  4. Institut Curie, Department of Translational Research, RPPA platform, PSL Research University, Paris, France

    Bérengère Ouine, Aurélie Cartier & Leanne de Koning

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  1. Chamseddine Ben Brahim
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  2. Charlotte Courageux
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Contributions

Study conception and design: PL; Conceived and Designed Experiments CBB, PL.

Conducted experiments, analyzed and interpreted data/: CBB, CC, A J, BO, AC, PdlG, LdK, PL; Writing - draft preparation, review and editing: PdlG, LdK, PL; Supervision: PdlG, LdK, PL; Funding acquisition: PL.

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Correspondence to Pascale Leroy.

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This study was performed in line with the principles of the Declaration of Helsinki. Approval was obtained from the ethics committee of Paris Nord, IRB 00006477 Paris 7 University, France.

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Supplementary Information

Fig. S1

Bronchial epithelial progenitors express Slug in their nuclei. Primary bronchial epithelial basal cells from normal (a) or COPD (b) subjects were grown on filters and analyzed undifferentiated at confluence by fluorescent immunocytochemistry. Cells were fixed and labeled simultaneously with progenitor cell marker p63 (white) and Slug (green) antibodies and with Hoechst as a marker of nuclei (blue). Bars are 20 μm (PPTX 1836 kb)

Fig. S2

Bronchial epithelial progenitors co-express epithelial and mesenchymal markers. Primary bronchial epithelial basal cells, normal and COPD, were grown on filters at confluence and either analyzed undifferentiated (a) or changed to ALI culture to induce differentiation, without TGF-β or in presence of 1 ng/ml of TGF-β and analyzed at day 6 of ALI culture for mRNA expression (b). RNA were extracted from normal and COPD cells and analyzed by RT-qPCR to determine the mRNA levels of KRT5 gene, an epithelial cytoskeletal marker and ACTA 2 and Vim genes, mesenchymal cytoskeletal markers.. GAPDH was used to normalize cDNA amounts between samples and results were calculated as a ratio on GAPDH. Data are for n ≥ 11 and experiments were done at least in duplicate. (a) Results shown are log2 (ratio on GAPDH) and are presented as a scatter plot with the mean ±SD. ns: non significant. (b) Results are presented as the fold-change induced by TGF-β on mRNA expression with mean ±SEM, and compare normal and COPD cells. Statistical significance is at P value < 1.00E-03 *** as indicated, ns is non significan (PPTX 129 kb)

Fig. S3

Slug and E-cad/CDH1 mRNA levels in normal and COPD bronchial epithelial progenitors in presence of TGF-β. Primary bronchial epithelial basal cells, normal and COPD, were grown on filters and at confluence changed to ALI culture to induce differentiation in presence of 1 ng/ml of TGF-β. Cells were analyzed at day 6 of ALI culture for Slug (a) and E-cad/CDH1 (b) mRNA expression. RNA were extracted from normal and COPD cells and analyzed by RT-qPCR. GAPDH was used to normalize cDNA amounts between samples and results were calculated as a ratio on GAPDH. Data shown represent the mean for n ≥ 11, and experiments were done at least in duplicate. Statistical significance is at P value < 5.00E-02. ns: non significant (PPTX 87 kb)

Fig. S4

Bronchial epithelial progenitors express Slug in their nuclei and co-express E-cadherin with or without TGF-β. Normal (a, b) or COPD (c, d) primary bronchial epithelial basal cells were grown on filters and at confluence changed to ALI culture to induce differentiation, without TGF-β (a, c) or in presence of 1 ng/ml of TGF-β (b, d). Cells were analyzed at day 6 of ALI culture by fluorescent immunocytochemistry. Cells were fixed and labeled simultaneously with Slug (green) and E-cad (red) antibodies and with Hoechst as a marker of nuclei (blue). Bars are 20 μm (PPTX 3593 kb)

Fig. S5

Effect of Slug knockdown on Slug mRNA and protein levels in normal and COPD bronchial epithelial progenitors. Normal and COPD bronchial epithelial basal/progenitor cells were transduced in transwell inserts with shRNA lentiviral particles corresponding to either a SNAI2/Slug specific sequence or control non-targeting sequences. At day 4 post-transduction, cells were changed to ALI conditions and at day 6 post-transduction cells were analyzed for mRNA or for protein expression. RNA or proteins lysates were prepared from normal and COPD cells and analyzed respectively by RT-qPCR (a) and by Western blot (b). For RT-qPCR analysis, GAPDH was used to normalize cDNA amounts between samples and results were calculated as a ratio on GAPDH. Results are presented as the fold-change induced by shRNA with SNAI2/Slug specific sequence on Slug mRNA expression (a) or Slug protein expression (b) with mean ±SEM. Data are for n ≥ 4. Statistical significance is at P value < 5.00E-02 *, < 1.00E-02 ** or < 1.00E-03 *** as indicated (PPTX 119 kb)

Fig. S6

Comparison of TGF-β effect on the expression of proliferation-related genes between normal and COPD bronchial epithelial progenitors. Primary bronchial epithelial basal cells, normal and COPD, were grown on filters and analyzed undifferentiated at confluence or grown on filters and at confluence changed to ALI culture to induce differentiation, without TGF-β or in presence of 1 ng/ml of TGF-β. Cells were analyzed at day 6 of ALI culture for mRNA expression. RNA were prepared from normal and COPD cells and analyzed respectively by RT-qPCR. GAPDH was used to normalize cDNA amounts between samples and results were calculated as a ratio on GAPDH. Data shown are for n ≥ 11. Results are presented as the fold-change induced by TGF-β on mRNA expression with mean ±SEM. Statistical significance is at P value < 1.00E-03 *** as indicated (PPTX 270 kb)

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Ben Brahim, C., Courageux, C., Jolly, A. et al. Proliferation Genes Repressed by TGF-β Are Downstream of Slug/Snail2 in Normal Bronchial Epithelial Progenitors and Are Deregulated in COPD. Stem Cell Rev and Rep 17, 703–718 (2021). https://doi.org/10.1007/s12015-021-10123-z

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