Limits...
Remote activation of the Wnt/β-catenin signalling pathway using functionalised magnetic particles.

Rotherham M, El Haj AJ - PLoS ONE (2015)

Bottom Line: Using this strategy, we determined the effects of mechano-stimulation of the Wnt Frizzled receptor on Wnt pathway activation in hMSC.We observed an increase in reporter activity after treatment with Fz-MNP and this effect was enhanced after mechano-stimulation using the magnetic array.Finally, the gene expression profiles of stress response genes were found to be similar when cells were treated with recombinant Wnt-3A or Fz-MNP.

View Article: PubMed Central - PubMed

Affiliation: Institute for Science and Technology in Medicine, Keele University, Stoke-on-Trent, United Kingdom.

ABSTRACT
Wnt signalling pathways play crucial roles in developmental biology, stem cell fate and tissue patterning and have become an attractive therapeutic target in the fields of tissue engineering and regenerative medicine. Wnt signalling has also been shown to play a role in human Mesenchymal Stem Cell (hMSC) fate, which have shown potential as a cell therapy in bone and cartilage tissue engineering. Previous work has shown that biocompatible magnetic nanoparticles (MNP) can be used to stimulate specific mechanosensitive membrane receptors and ion channels in vitro and in vivo. Using this strategy, we determined the effects of mechano-stimulation of the Wnt Frizzled receptor on Wnt pathway activation in hMSC. Frizzled receptors were tagged using anti-Frizzled functionalised MNP (Fz-MNP). A commercially available oscillating magnetic bioreactor (MICA Biosystems) was used to mechanically stimulate Frizzled receptors remotely. Our results demonstrate that Fz-MNP can activate Wnt/β-catenin signalling at key checkpoints in the signalling pathway. Immunocytochemistry indicated nuclear localisation of the Wnt intracellular messenger β-catenin after treatment with Fz-MNP. A Wnt signalling TCF/LEF responsive luciferase reporter transfected into hMSC was used to assess terminal signal activation at the nucleus. We observed an increase in reporter activity after treatment with Fz-MNP and this effect was enhanced after mechano-stimulation using the magnetic array. Western blot analysis was used to probe the mechanism of signalling activation and indicated that Fz-MNP signal through an LRP independent mechanism. Finally, the gene expression profiles of stress response genes were found to be similar when cells were treated with recombinant Wnt-3A or Fz-MNP. This study provides proof of principle that Wnt signalling and Frizzled receptors are mechanosensitive and can be remotely activated in vitro. Using magnetic nanoparticle technology it may be possible to modulate Wnt signalling pathways and thus control stem cell fate for therapeutic purposes.

No MeSH data available.


Related in: MedlinePlus

Frizzled-MNP promote β-catenin activation and mobilisation.Fluorescent images showing localisation of active β-catenin (Green) after 24h, DAPI was used to visualise cell nuclei (Blue). Non-treated cells (A) showed negligible β-catenin localisation. Cells treated IgG-MNP or RGD-MNP (A) resulted in a small non-significant increase in nuclear localisation, and addition of magnetic field (B) had no observable effect. Cells treated with magnetic field alone (B) showed a moderate increase in β-catenin localisation. Treatment with Anti-frizzled magnetic nanoparticles (Fz-MNP) without magnet (A) showed notable nuclear localisation with an added effect when used in conjunction with magnetic field (B). Treatment with Wnt-conditioned media (A) also showed notable nuclear β-catenin after 24h. Representative images, n = 3, scale bar = 50μm. Quantification of nuclear pixel intensity (C) indicated levels of nuclear (active) β-catenin. Treatment with magnetic field alone, IgG-MNP and RGD-MNP (with or without magnetic field) all caused similar increases in levels of nuclear β-catenin. Fz-MNP and Wnt-CM both increased β-catenin mobilisation to similar levels and an added effect was observed when Fz-MNP were used in conjunction with magnetic field. Average pixel intensities shown, n = 3, error bars represent SEM, * denotes p<0.05
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4363733&req=5

pone.0121761.g004: Frizzled-MNP promote β-catenin activation and mobilisation.Fluorescent images showing localisation of active β-catenin (Green) after 24h, DAPI was used to visualise cell nuclei (Blue). Non-treated cells (A) showed negligible β-catenin localisation. Cells treated IgG-MNP or RGD-MNP (A) resulted in a small non-significant increase in nuclear localisation, and addition of magnetic field (B) had no observable effect. Cells treated with magnetic field alone (B) showed a moderate increase in β-catenin localisation. Treatment with Anti-frizzled magnetic nanoparticles (Fz-MNP) without magnet (A) showed notable nuclear localisation with an added effect when used in conjunction with magnetic field (B). Treatment with Wnt-conditioned media (A) also showed notable nuclear β-catenin after 24h. Representative images, n = 3, scale bar = 50μm. Quantification of nuclear pixel intensity (C) indicated levels of nuclear (active) β-catenin. Treatment with magnetic field alone, IgG-MNP and RGD-MNP (with or without magnetic field) all caused similar increases in levels of nuclear β-catenin. Fz-MNP and Wnt-CM both increased β-catenin mobilisation to similar levels and an added effect was observed when Fz-MNP were used in conjunction with magnetic field. Average pixel intensities shown, n = 3, error bars represent SEM, * denotes p<0.05

Mentions: To determine if Fz-MNP were causing Wnt signalling activation downstream of Frizzled, the mobilisation and nuclear localisation of the intracellular messenger β-catenin was investigated. β-catenin localisation was studied in hMSC after treatment with Fz-MNP with and without magnetic field or Wnt-CM. Fig. 4A shows that the non-treated cells displayed negligible β-catenin nuclear localisation after 24h. When cells were treated with anti-Fz MNP without magnetic field, clear nuclear localisation of β-catenin was observed and pixel intensity analysis of the cell nuclei confirmed a significant increase in nuclear localisation compared to non-treated control. Magnetic field stimulation of Fz-MNP labelled cells resulted in a further increase in β-catenin mobilisation (Fig. 4B). Pixel intensity analysis (Fig. 4C) also confirmed a significant increase in nuclear staining compared to cells + magnet control. Cells treated with Wnt-CM (Fig. 4A) showed noticeable nuclear localisation after 24h which was significant over non-treated control according to pixel intensity analysis. Control particles IgG-MNP and RGD-MNP (Fig. 4A) caused minor increases in β-catenin activation and no additive effect was observed with addition of magnetic field (Fig. 4B).


Remote activation of the Wnt/β-catenin signalling pathway using functionalised magnetic particles.

Rotherham M, El Haj AJ - PLoS ONE (2015)

Frizzled-MNP promote β-catenin activation and mobilisation.Fluorescent images showing localisation of active β-catenin (Green) after 24h, DAPI was used to visualise cell nuclei (Blue). Non-treated cells (A) showed negligible β-catenin localisation. Cells treated IgG-MNP or RGD-MNP (A) resulted in a small non-significant increase in nuclear localisation, and addition of magnetic field (B) had no observable effect. Cells treated with magnetic field alone (B) showed a moderate increase in β-catenin localisation. Treatment with Anti-frizzled magnetic nanoparticles (Fz-MNP) without magnet (A) showed notable nuclear localisation with an added effect when used in conjunction with magnetic field (B). Treatment with Wnt-conditioned media (A) also showed notable nuclear β-catenin after 24h. Representative images, n = 3, scale bar = 50μm. Quantification of nuclear pixel intensity (C) indicated levels of nuclear (active) β-catenin. Treatment with magnetic field alone, IgG-MNP and RGD-MNP (with or without magnetic field) all caused similar increases in levels of nuclear β-catenin. Fz-MNP and Wnt-CM both increased β-catenin mobilisation to similar levels and an added effect was observed when Fz-MNP were used in conjunction with magnetic field. Average pixel intensities shown, n = 3, error bars represent SEM, * denotes p<0.05
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4363733&req=5

pone.0121761.g004: Frizzled-MNP promote β-catenin activation and mobilisation.Fluorescent images showing localisation of active β-catenin (Green) after 24h, DAPI was used to visualise cell nuclei (Blue). Non-treated cells (A) showed negligible β-catenin localisation. Cells treated IgG-MNP or RGD-MNP (A) resulted in a small non-significant increase in nuclear localisation, and addition of magnetic field (B) had no observable effect. Cells treated with magnetic field alone (B) showed a moderate increase in β-catenin localisation. Treatment with Anti-frizzled magnetic nanoparticles (Fz-MNP) without magnet (A) showed notable nuclear localisation with an added effect when used in conjunction with magnetic field (B). Treatment with Wnt-conditioned media (A) also showed notable nuclear β-catenin after 24h. Representative images, n = 3, scale bar = 50μm. Quantification of nuclear pixel intensity (C) indicated levels of nuclear (active) β-catenin. Treatment with magnetic field alone, IgG-MNP and RGD-MNP (with or without magnetic field) all caused similar increases in levels of nuclear β-catenin. Fz-MNP and Wnt-CM both increased β-catenin mobilisation to similar levels and an added effect was observed when Fz-MNP were used in conjunction with magnetic field. Average pixel intensities shown, n = 3, error bars represent SEM, * denotes p<0.05
Mentions: To determine if Fz-MNP were causing Wnt signalling activation downstream of Frizzled, the mobilisation and nuclear localisation of the intracellular messenger β-catenin was investigated. β-catenin localisation was studied in hMSC after treatment with Fz-MNP with and without magnetic field or Wnt-CM. Fig. 4A shows that the non-treated cells displayed negligible β-catenin nuclear localisation after 24h. When cells were treated with anti-Fz MNP without magnetic field, clear nuclear localisation of β-catenin was observed and pixel intensity analysis of the cell nuclei confirmed a significant increase in nuclear localisation compared to non-treated control. Magnetic field stimulation of Fz-MNP labelled cells resulted in a further increase in β-catenin mobilisation (Fig. 4B). Pixel intensity analysis (Fig. 4C) also confirmed a significant increase in nuclear staining compared to cells + magnet control. Cells treated with Wnt-CM (Fig. 4A) showed noticeable nuclear localisation after 24h which was significant over non-treated control according to pixel intensity analysis. Control particles IgG-MNP and RGD-MNP (Fig. 4A) caused minor increases in β-catenin activation and no additive effect was observed with addition of magnetic field (Fig. 4B).

Bottom Line: Using this strategy, we determined the effects of mechano-stimulation of the Wnt Frizzled receptor on Wnt pathway activation in hMSC.We observed an increase in reporter activity after treatment with Fz-MNP and this effect was enhanced after mechano-stimulation using the magnetic array.Finally, the gene expression profiles of stress response genes were found to be similar when cells were treated with recombinant Wnt-3A or Fz-MNP.

View Article: PubMed Central - PubMed

Affiliation: Institute for Science and Technology in Medicine, Keele University, Stoke-on-Trent, United Kingdom.

ABSTRACT
Wnt signalling pathways play crucial roles in developmental biology, stem cell fate and tissue patterning and have become an attractive therapeutic target in the fields of tissue engineering and regenerative medicine. Wnt signalling has also been shown to play a role in human Mesenchymal Stem Cell (hMSC) fate, which have shown potential as a cell therapy in bone and cartilage tissue engineering. Previous work has shown that biocompatible magnetic nanoparticles (MNP) can be used to stimulate specific mechanosensitive membrane receptors and ion channels in vitro and in vivo. Using this strategy, we determined the effects of mechano-stimulation of the Wnt Frizzled receptor on Wnt pathway activation in hMSC. Frizzled receptors were tagged using anti-Frizzled functionalised MNP (Fz-MNP). A commercially available oscillating magnetic bioreactor (MICA Biosystems) was used to mechanically stimulate Frizzled receptors remotely. Our results demonstrate that Fz-MNP can activate Wnt/β-catenin signalling at key checkpoints in the signalling pathway. Immunocytochemistry indicated nuclear localisation of the Wnt intracellular messenger β-catenin after treatment with Fz-MNP. A Wnt signalling TCF/LEF responsive luciferase reporter transfected into hMSC was used to assess terminal signal activation at the nucleus. We observed an increase in reporter activity after treatment with Fz-MNP and this effect was enhanced after mechano-stimulation using the magnetic array. Western blot analysis was used to probe the mechanism of signalling activation and indicated that Fz-MNP signal through an LRP independent mechanism. Finally, the gene expression profiles of stress response genes were found to be similar when cells were treated with recombinant Wnt-3A or Fz-MNP. This study provides proof of principle that Wnt signalling and Frizzled receptors are mechanosensitive and can be remotely activated in vitro. Using magnetic nanoparticle technology it may be possible to modulate Wnt signalling pathways and thus control stem cell fate for therapeutic purposes.

No MeSH data available.


Related in: MedlinePlus