Adhesion of basal keratinocytes to the underlying extracellular matrix (ECM) plays a key role in the control of skin homeostasis and response to injury. Integrin receptors indirectly link the extracellular matrix to the cell cytoskeleton through large protein complexes called focal adhesions (FA). FA also function as intracellular biochemical signalling platforms to enable cells to respond to changing extracellular cues. The α4β1 and α9β1 integrins are both expressed in basal keratinocytes, share some common ECM ligands and have been shown to promote wound healing in vitro and in vivo. However, their roles in maintaining epidermal homeostasis and relative contributions to pathological processes in the skin remain unclear. To further define the role of these integrins in epithelial cell migration, monolayers of immortalised human keratinocytes were treated with α4β1-specific or dual α4β1/α9β1 integrin inhibitors. Analysis of time-lapse live cell and fixed cell images revealed that inhibition of α4β1 and α9β1 integrins increased keratinocyte proliferation and induced a dramatic change in cytoskeletal remodelling and FA rearrangement. Moreover, α4β1/α9β1 inhibition reduced collective cell migration compared to control cells. To define the mechanisms by which α4β1/α9β1 control migration, we conducted a screen and identified Mitogen and Stress Activated Kinase 1 (MSK1) as a key hit that was increased in activity upon integrin inhibition. Further analysis revealed that MSK1, reported to be a predominantly nuclear-localised kinase, was restricted to the cytoplasm of keratinocyte monolayers, and inhibition of α4β1 or α4β1/α9β1 disrupted this localisation. Moreover, α4β1/α9β1 inhibition also led to increased ERK1/2 phosphorylation. As ERK1/2 promotes phosphorylation of MSK1, this data suggests that α4β1/α9β1 integrins may suppress local ERK1/2 activity to control migration through the regulation of downstream kinases including MSK1. Therefore, this thesis demonstrates the roles of α4β1 and α9β1 integrins in the regulation of keratinocyte homeostasis, and their potential roles in controlling proliferation and migration in response to damage cues.
Analysing contributions of α4 and α9 integrin to epithelial cell migration
Hight-Warburton, W. (Author). 1 Mar 2021
Student thesis: Doctoral Thesis › Doctor of Philosophy