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

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.

Fig 1. Magnetic Force Bioreactor.

Image of the Magnetic Force Bioreactor used in magnetic stimulation experiments. Culture plates are situated on the plate holder above the magnetic array which oscillates vertically beneath the culture plates. The movement parameters for the array are computer controlled.

Fig 2. Frizzled antibody reduces Fz-MNP binding.

Immunofluorescence images of cells labelled with Fz-MNP (A, C, E, G) or pre-blocked with anti-frizzled antibody before labelling with Fz-MNP (B, D, F, H). Cytoskeleton was visualised using Phalloidin (A, B), Anti-Dextran (C, D) was used to visualise Fz-MNP. Cell nuclei are shown by DAPI staining (E, F). Merged images are shown in G, H. Scale bar = 50μm. Images representative of n = 3.

Fig 3. Fz-MNP signal through an LRP Independent mechanism.

Treatment of hMSC with Frizzled-particles (Fz-MNP) did not result in phosphorylation of Wnt co-receptor LRP6. In contrast treatment with Wnt conditioned media (Wnt-CM) resulted in clear LRP6 phosphorylation which was blocked using the Wnt inhibitor Dickkopf related protein 1 (Dkk1). Non-treated cells displayed a basal level of Wnt co-receptor LRP5 phosphorylation (B). Treatment with Frizzled particles (Fz-MNP) or Wnt conditioned media (Wnt-CM) had no observable effect on the phosphorylation levels of LRP5. Black lines denote none adjacent lanes.

Fig 4. 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

Fig 5. Anti-Frizzled particles activate a Wnt TCF/LEF luciferase reporter.

TCF/LEF reporter activity from transiently transfected hMSC 6h and 24h after treatment. Magnetic field alone (hMSC + Mag) increased reporter activity at both time points (not significant) (A, B). Treatment with Frizzled particles (Fz-MNP) without magnetic field caused a noticeable increase in reporter activity over both time-points (trend only). An added significant increase in reporter activity was observed over both time-points when magnetic field was applied. Addition of the Wnt signalling blocker Dkk1 which inhibits LRP5/6 activation failed to prevent reporter activation by Fz-MNP. However activation was successfully blocked using the TCF/LEF inhibitor iCRT14, which inhibits Wnt signalling downstream of Frizzled. Treatment with Wnt-Conditioned Media increased reporter activity to a similar level as Fz-MNP (without magnet field) after 6h (A), with maximum activity observed after 24h (B). This effect was blocked using Dkk1 or iCRT14 with reporter activity being reduced to basal levels. Control particles coated with either Rabbit-IgG (IgG-MNP) or RGD peptide (RGD-MNP) caused no increase in reporter activity at 6h (C) or 24h (D) with or without magnetic field. Values represent mean fold change in luciferase activity relative to cells only with luciferase activity normalised to total protein. Error bars represent SEM, n = 4, * denotes p<0.05, # denotes p ≥ 0.05

Fig 6. Anti-Frizzled MNP alter expression of Mechanosensitive genes.

Gene expression analysis of mechano responsive genes (NF-κB, c-Myc, Cox 2) in response to anti-frizzled particles (Fz-MNP) or control particles (Trek-MNP) and magnetic field stimulation was evaluated using quantitative real-time PCR after 1h and 3h treatment. NF-κB gene expression (A) was increased by Trek-MNP (compared to cells + magnet control) after 1h. Magnetic field treatment alone also increased NF-κB expression (compared to cells only control) after 3h. Treatment with Fz-MNP or Wnt 3A both showed similar but minor shifts in NF-κB gene expression. c-Myc expression (B) was increased after 1h treatment with magnetic field treatment alone (cells + magnet) with an added increase when used in conjunction with Trek-MNP. Treatment with Fz-MNP or Wnt 3A again both showed similar but negligible shifts in c-Myc expression. COX 2 gene expression (C) was shown to be significantly increased after 1h by magnetic field treatment alone but a decrease in expression was observed after 3h. Treatment with Fz-MNP (without magnet) or Wnt 3A both caused significant decreases in COX 2 expression after 3h treatment. Treatment with Fz-MNP (+magnet) was shown to significantly increase COX 2 expression when compared to Cells + magnet control after 3h. Figures show mean fold change in gene expression normalised to GAPDH, Error bars represent SEM, n = 4, ANOVA p < 0.05 for all genes, * denotes p <0.05.