Trafficking and signalling of Syndecan-4 during wound healing

Wound healing defects affect over 2.2 million patients and cost £4-5 billion per year in the UK. Improper healing often progresses to chronic ulceration with excruciating pain, tissue necrosis and frequently limb amputation. Upon injury, syndecan-4 receptor binds fibronectin from damaged blood vessel activating PKCα and Rac1 at the leading edge of fibroblasts mediating cell polarisation, cytoskeletal rearrangement, redistribution of receptors and migration to wound site. Syndecan-4 wild type MEFs form vinculin-containing focal adhesion when stimulated with heparin-binding fragment of fibronectin that forms the syndecan-4 ligand. In contrast, syndecan-4 knockout MEFs fail to respond, supporting previous reports where knockout mice suffered delayed healing due to migration defect establishing the importance of these molecules during the healing response. Syndecan-4 enables fibroblast to detect changes in the environment and regulates the redistribution of integrin to provide a physical link between ECM and cytoskeletal contractile machinery. Syndecan-4 regulation is still unclear hence, understanding syndecan-4 trafficking will be a key step in elucidating how fibroblasts polarise and migrate in response to wounding. Measuring syndecan-4 uptake in fibroblasts using biotinylated and fluorophore-conjugated antibody demonstrated involvement and colocalisation of the receptor with caveolin and clathrin within 30 minutes of internalisation. Syndecan-4 receptor engagement activates PKCα to mediate receptor downstream signalling including Rac1 activation and localisation to the leading edge of migrating fibroblasts. The role of PKCα on syndecan-4 uptake was tested by RNAi-mediated knockdown of PKCα and BIM-I treated cells demonstrating attenuated receptor uptake suggesting that PKCα activity is required for endocytosis. Mutation of the PKCα-binding motif of syndecan-4 blocked syndecan-4 endocytosis, demonstrating that it self-regulates. Syndecan-4 fab fragment failed to trigger receptor uptake, despite binding, suggesting that clustering is required for receptor internalisation. In conclusion, data demonstrates that syndecan-4 clustering is required for caveolin- and clathrin-dependant endocytosis of syndecan-4 in a PKCα-dependant manner.