Date Published: March 17, 2015
Publisher: Public Library of Science
Author(s): Michael Rotherham, Alicia J. El Haj, Shao-Jun Tang.
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.
Wnt signalling is a complex pathway involved in the regulation of a range of biological processes ranging from cell proliferation and differentiation to embryogenesis, tissue formation and regulation of stem cell niches  . In humans Wnt proteins consist of a class of nineteen evolutionary conserved, glycosylated and lipid modified proteins each harbouring a cysteine rich domain  . The primary receptor for Wnt ligands are the Frizzleds, a family of ten G-protein like 7- transmembrane spanning receptors . Frizzleds have an extended N-terminal region harbouring a cysteine rich domain (CRD) which is required for Wnt reception   . In Canonical Wnt/β-catenin signalling, Wnt proteins interact with a receptor complex comprised of Frizzled (Fz)/Low density lipoprotein (LDL) receptor-related protein (LRP) located on the cell membrane   . When activated by Wnt ligands, LRP becomes phosphorylated at multiple sites. The activated Fz/LRP receptor complex recruits Axin to the cell membrane  ; this causes the sequestration of several other intracellular proteins required for Wnt signalling modulation, including glycogen synthase kinase-3β (GSK-3), Dishevelled (Dsh) and Adenomatous Polyposis Coli (APC). In the absence of an external Wnt signal GSK-3, Dsh and APC form a destruction complex that regulates the cytoplasmic pool of the transcriptional regulator β-catenin  by successive phosphorylation which marks β-catenin for proteasome degradation  . In the presence of a Wnt signal the destruction complex dissociates and is deactivated; as a result active β-catenin accumulates in the cytoplasm and nucleus. Nuclear β-catenin acts as a transcriptional regulator and interacts with the lymphoid enhancer-binding factor 1/T-cell specific transcription factor (LEF/TCF) family of transcription factors which bind to and activate Wnt responsive genes with TCF/LEF binding sites .
Wnt signalling is a crucial pathway controlling stem cell behaviour and in recent years has become an attractive target for modulation with potential applications in stem cell therapies. This study has demonstrated the feasibility of using anti-body functionalised magnetic nanoparticles to specifically target and stimulate Frizzled 2 receptors expressed by hMSC for the activation of Wnt/β-catenin signalling pathways.
Wnt signalling pathways are important for the regulation of cell behaviour and there is increasing interest in the development of modulators of Wnt signalling. These tools may have therapeutic potential in fields such as stem cell science, mammalian development, cell and tissue engineering and cancer. One way of controlling cell signalling is by using bio-functionalised MNP. To date no one has attempted to locally target and stimulate Wnt receptors in order to modulate Wnt signalling. Our work has shown that it is possible to tag Frizzled receptors on hMSC with antibody-functionalised nano-particles and use an oscillating magnetic field to impart focused mechanical stimulation of Frizzled receptors. This strategy has enabled the unconventional activation of Wnt signalling pathways in hMSC. Immunocytochemistry has demonstrated the targeting specificity of Fz-MNP with the use of blocking antibodies. Western blotting has indicated that Frizzled receptor and subsequent Wnt signalling activation with Fz-MNP is independent of LRP. Immunofluorescence studies showed mobilisation of the Wnt signalling messenger β-catenin in response to Fz-MNP treatment and activation of a Wnt signalling reporter construct. Gene expression analysis has shown differential expression of stress response genes in response to Fz-MNP and Wnt treatment. Taken together, these results suggest that Fz-MNP and Wnts are acting via different mechanisms to activate Wnt/β-catenin signalling. The mechanism behind signal activation through Fz-MNP and the effects on hMSC fate and differentiation requires further investigation. Also the remote activation of other Frizzled receptors and co-receptors for the modulation of Wnt pathways using MNP technology should be investigated. The development of this technology raises the possibility of remotely controlling Wnt signalling and consequently the control of stem cell behaviour.