The influence of cx43 protein on keratinocyte migration

The gap junction protein, Cx43, naturally down regulates in keratinocytes at the edge of a normal acute wound as they become migratory. However, in diabetic skin and aged skin Cx43 up-regulates and migration fails until Cx43 down regulation occurs (either naturally or following antisense treatment). Pilot data, from non-diabetic human chronic wounds, shows that Cx43 protein levels are very high in the hyper-thickened wound edge and no keratinocyte migration is taking place. Such wounds are extremely debilitating and have a major negative impact on lifestyle of typically elderly sufferers. Work from the Becker lab at UCL has shown that transiently decreasing the level of Cx43 protein at a wound site, by application of an antisense gel, accelerates the normal rate of wound healing and also rescues that of diabetic wounds. This technology is being developed by CoDa Therapeutics Inc. (CoDa), our industrial sponsor, and has been taken into clinical trials. However, the cell biology behind the action of Cx43 on the rate of healing is still to be fully understood. This project aims to enhance our understanding of the influence of Cx43 in relation to keratinocyte migration, an event that is of vital importance if chronic wounds are to be induced to heal. We will use in vivo and in vitro wound healing to examine the effects of Cx43 protein expression on keratinocyte migration. Using our in vitro models of wound healing we will image live cell migration dynamics of keratinocytes whose Cx43 expression has been up or down regulated by pharmacological treatment or transfection with wild type Cx43, dominant negative Cx43 or Cx43shRNA. Intracellular dye injection in cell cultures, will be used to monitor any changes that take place in cell-cell communication of leading edge cells and those that follow. We have recently shown that Cx43 expression affects the expression of several cytoskeletal and adhesion proteins that bind to it. We will therefore also examine the effects of the presence or absence of Cx43 protein on the distribution of adhesion complex proteins such as ZO-1, N-Cadherin, Beta-Catenin as well as associated cytoskeletal proteins actin and tubulin. Pilot data suggest there are significant changes in these protein levels and distributions. In order to demonstrate that these observations also occur in vivo we will use animal models of wound healing where we will also carry out adhesion complex and cytoskeletal protein immunostaining studies on tissue sections from normal and diabetic rodent wounds (that have elevated Cx43 levels and retarded healing) which have been treated with Cx43 antisense or vehicle control. In addition we now have a bio-bank of samples of a variety of human normal tissue, and acute human and chronic wound tissue, available to us in which we would be able to carry out immunostaining for connexins, adhesion complexes and the cytoskeleton in order to draw correlations with their migratory state. Should time permit, we are well placed within the Centre for Cell and Molecular Dynamics Imaging Facility to start to examine the effects of Cx43 protein expression on dynamics of cytoskeletal components labelled with GFP or RFP. In addition it would be possible to image the effects cell migration and the interaction of Rac and Rho as shown by FRET pair imaging. The biological side of this project should provide the student with experience in a wide variety of modern cell biology and live cell imaging techniques as well as provide useful information on the cell biology of wound healing in relation to connexins. Exposure to the company should give experience of how a young and dynamic biotech company functions.