Multi-walled carbon nanotubes directly induce epithelial-mesenchymal transition in human bronchial epithelial cells via the TGF-β-mediated Akt/GSK-3β/SNAIL-1 signalling pathway

Our plain-English summary of Multi-walled carbon nanotubes directly induce epithelial-mesenchymal transition in human bronchial epithelial cells via the TGF-β-mediated Akt/GSK-3β/SNAIL-1 signalling pathway, by M. Polimeni, G.R. Gulino, E. Gazzano, et al (Particle and Fibre Toxicology, 2016).

The study

• Authors: M. Polimeni, G.R. Gulino, E. Gazzano, J. Kopecka, A. Marucco, I. Fenoglio, F. Cesano, L. Campagnolo, A. Magrini, A. Pietroiusti, D. Ghigo, E. Aldieri
• Journal: Particle and Fibre Toxicology
• Published: 2016;13(1):27
• Cited by: 63 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Multi-walled carbon nanotubes (MWCNT) are currently under intense toxicological investigation due to concern on their potential health effects. Current in vitro and in vivo data indicate that MWCNT exposure is strongly associated with lung toxicity (inflammation, fibrosis, granuloma, cancer and airway injury) and their effects might be comparable to asbestos-induced carcinogenesis. Although fibrosis is a multi-origin disease, epithelial-mesenchymal transition (EMT) is recently recognized as an important pathway in cell transformation. It is known that MWCNT exposure induces EMT through the activation of the TGF-β/Smad signalling pathway thus promoting pulmonary fibrosis, but the molecular mechanisms involved are not fully understood. In the present work we propose a new mechanism involving a TGF-β-mediated signalling pathway.

Methods: Human bronchial epithelial cells were incubated with two different MWCNT samples at various concentrations for up to 96 h and several markers of EMT were investigated. Quantitative real time PCR, western blot, immunofluorescent staining and gelatin zymographies were performed to detect the marker protein alterations. ELISA was performed to evaluate TGF-β production. Experiments with neutralizing anti-TGF-β antibody, specific inhibitors of GSK-3β and Akt and siRNA were carried out in order to confirm their involvement in MWCNT-induced EMT. In vivo experiments of pharyngeal aspiration in C57BL/6 mice were also performed. Data were analyzed by a one-way ANOVA with Tukey's post-hoc test.

Results: Fully characterized MWCNT (mean length < 5 μm) are able to induce EMT in an in vitro human model (BEAS-2B cells) after long-term incubation at sub-cytotoxic concentrations. MWCNT stimulate TGF-β secretion, Akt activation and GSK-3β inhibition, which induces nuclear accumulation of SNAIL-1 and its transcriptional activity, thus contributing to switch on the EMT program. Moreover, a significant increment of nuclear β-catenin - due to E-cadherin repression and following translocation to nucleus - likely reinforces signalling for EMT promotion. In vivo results supported the occurrence of pulmonary fibrosis following MWCNT exposure.

Conclusions: We demonstrate a new molecular mechanism of MWCNT-mediated EMT, which is Smad-independent and involves TGF-β and its intracellular effectors Akt/GSK-3β that activate the SNAIL-1 signalling pathway. This finding suggests potential novel targets in the development of therapeutic and preventive approaches.

In plain English

This laboratory study tested two multi-walled carbon nanotube (MWCNT) samples: pristine MWCNT (pMWCNT, physicochemically similar to MITSUI-7, classified by IARC as possibly carcinogenic to humans, Group 2B) and a ground sample shortened to a mean length of 1.12 micrometres (MWCNTg). Human bronchial epithelial BEAS-2B cells were exposed to 5.5, 11, 22 and 44 micrograms per millilitre for up to 96 hours, and C57BL/6 mice received 120 micrograms by pharyngeal aspiration. After 96 hours, LDH leakage rose significantly only at the two highest concentrations (14.9 per cent in controls versus 29.2 and 31.9 per cent at 22 and 44 micrograms per millilitre of MWCNTg). Both samples changed cell shape, lowered E-cadherin and raised vimentin, alpha-SMA, fibronectin, and MMP-2 and MMP-9 activity. With MWCNTg, TGF-beta secretion, Akt phosphorylation, GSK-3beta phosphorylation and nuclear SNAIL-1 increased; co-incubation with inhibitors or siRNA against TGF-beta, Akt or GSK-3beta completely reversed the nuclear accumulation of SNAIL-1 and partially restored E-cadherin. In mice, BAL TGF-beta and lung hydroxyproline increased. The authors concluded that MWCNTg promote epithelial-mesenchymal transition through the TGF-beta-mediated Akt/GSK-3beta/SNAIL-1 pathway, with the animal results supporting pulmonary fibrosis after MWCNT treatment.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: In vitro experiments in a human bronchial epithelial cell line, with supporting in vivo pharyngeal aspiration experiments in mice
• Who was studied: Human bronchial epithelial BEAS-2B cells (ATCC) in culture, and eight week-old male C57BL/6 mice, at least 4 animals per experimental group, studied at 15, 30 and 60 days after exposure
• Exposure: Pristine MWCNT (pMWCNT, similar to MITSUI-7), ground MWCNT (MWCNTg, mean length 1.12 micrometres, mean diameter 67 ± 2 nm) and Fe-depleted MWCNTg; cells at 5.5, 11, 22 and 44 micrograms per millilitre up to 96 h; mice given 120 micrograms by pharyngeal aspiration
• Main results:
  • In BEAS-2B cells at 96 h, LDH leakage was significantly increased only at the two highest MWCNTg concentrations: 14.9 per cent in control cells versus 29.2 per cent (22 micrograms per millilitre) and 31.9 per cent (44 micrograms per millilitre)
  • Both MWCNT samples raised vimentin and alpha-SMA mRNA concentration-dependently and repressed E-cadherin mRNA in BEAS-2B cells; the effect on mesenchymal markers was more evident with the pristine sample
  • Cells exposed to 44 micrograms per millilitre MWCNTg showed nuclear SNAIL-1 accumulation after 1 h with increased cytosolic GSK-3beta phosphorylation, and increased phosphorylated Akt after 30 min peaking at 2 h
  • Inhibiting GSK-3beta (5 micromolar SB216763) or using 10 nanomolar GSK-3beta siRNA prevented the nuclear increase of SNAIL-1 in MWCNTg-exposed cells
  • After 96 h co-incubation of MWCNTg with Akt or GSK-3beta siRNA or their specific inhibitors, nuclear SNAIL-1 accumulation was completely reversed and E-cadherin levels were only partially restored
  • Neutralizing anti-TGF-beta antibody (5 micrograms per millilitre) or TGF-beta siRNA (10 nanomolar) co-incubated for 96 h partially restored E-cadherin and completely reversed SNAIL-1 nuclear accumulation in MWCNTg-exposed cells
  • The authors took the antibody and siRNA experiments as confirming TGF-beta involvement in their cellular model
  • Extracellular MMP-2 and MMP-9 activity was significantly raised in the medium of cells exposed for 96 h to increasing concentrations of both MWCNT samples
  • In mice, BAL TGF-beta rose significantly by 15 days after pMWCNT, peaked at 30 days and remained significant at 60 days; MWCNTg produced a similar but less evident increase
  • Lung hydroxyproline content was significantly higher in mice treated with both MWCNT samples, with Sirius red staining more pronounced after pMWCNT
  • Fe3+ alone did not change cell morphology or epithelial markers, while Fe-depleted MWCNTg produced changes similar to iron-containing MWCNTg
• The authors' conclusion: The authors concluded that MWCNTg promote epithelial-mesenchymal transition through the TGF-beta-mediated Akt/GSK-3beta/SNAIL-1 pathway, and that in vivo experiments in C57BL/6 mice supported the development of pulmonary fibrosis after MWCNT treatment
• Limitations the authors state:
  • E-cadherin expression was only partly restored when the pathway was blocked, which the authors attribute to TGF-beta being just one of several possible mediators of MWCNT-induced EMT
  • The length distribution of the pristine MWCNT sample could not be measured quantitatively
  • The authors state that no relationship between human disease and occupational or environmental MWCNT exposure has been demonstrated
  • Further investigation is still required to clarify a possible crosstalk between beta-catenin and SNAIL-1 signalling in EMT induction
  • Some supporting observations in A549 cells are reported without shown data

Topics

Medical terms in this study, explained in our Lexicon: Inflammation, Fibrosis, Granuloma, Carcinogenesis, Pulmonary Fibrosis, Translocation.

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