Dissecting receptor crosstalk mechanisms that co-ordinate wound healing and drive scar formation

We will analyse crosstalk mechanisms between integrins and growth factor receptors (GFRs) that promote wound healing and drive scar formation.

TGF-B and integrin aVB6 cooperate to drive wound healing by inducing re-epithelialisation, myofibroblast activation, wound contraction and matrix remodelling. Integrin aVB6 activates TGF-B by binding and applying forces to latent TGF-B, inducing a structural change that releases the potent cytokine. Consequently, genetic deletion or inhibition of aVB6 or TGF-B leads to pronounced wound healing defects in vivo. However, despite the fundamental role of aVB6 in tissue regeneration, the molecular basis for aVB6-mediated wound repair is unresolved.
We found that: 1) aVB6 signalling complexes recruit a mucin-galectin-3 regulatory module; 2) MUC1-galectin-3 interaction regulates GFR clustering and trafficking. In vivo, MUC1 and galectin-3 promote wound repair and our iCASE partner has developed galectin-3-targeting drugs that inhibit TGF-B activity in in vivo models of fibrosis. These data suggest that aVB6, MUC1 and galectin-3 may co-operate to regulate TGF-B activation and aVB6-mediated epithelial cell migration.
MUC1 spatially-constrains integrin activation; the bulky glycoprotein funnels receptors into adhesions and applies compressive tension to promote integrin activation. This new paradigm of integrin activation leads to our hypothesis: Galectin-3 and MUC1 co-ordinate aVB6 clustering to drive TGF-B activation during wound repair.

Objectives & Experimental Approach
1) Determine impact of MUC1-galectin-3 interaction on aVB6 trafficking, ligand engagement and migration:
aVB6 trafficking will be analysed using biochemical endocytosis/recycling assays. aVB6 clustering and ligand-binding will be analysed by immunofluorescence using activation-reporting aVB6 antibodies. Haptotactic epithelial cell migration will be analysed in scratch wound assays and on fibrillar cell-derived matrices.

Contributions of MUC1 compressive tension or signalling will be analysed using bio-engineered mechanically-tuned glycoprotein-mimetics and MUC1 ectodomain/cytodomain mutants in skin epithelial cells. Involvement of MUC1-galectin-3 interaction, will be assessed in presence/absence of galectin-3 or galectin-3-targeting drugs. Role of aVB6 engagement will be determined using anti-aVB6 blocking antibodies.

2) Analyse whether MUC1-galectin-3 regulates aVB6-mediated force application and TGF-B activation:
Traction Force Microscopy (TFM) using latency-associated peptide as a ligand and TGF-B1 activity luciferse-reporter assays will demonstrate whether the biophysical and signalling properties of MUC1-galectin-3 regulate 1) application of aVB6-mediated mechanical forces on latent TGF-B, and 2) aVB6-dependent TGF-B activation.

3) Test whether MUC1-galectin-3 interaction co-ordinates aVB6 funnelling to drive TGF-B activation:
Topographical-Scanning Angle Interference Microscopy (T-SAIM); analysing membrane deformation using FRET reporters of MUC1 compression (compressive-strain gauge sensors) and aVB6 activation-reporting antibodies. Computational modelling will map compressive tension within individual MUC1 molecules, relative to active and inactive aVB6.