The Role of Endothelial Focal Adhesion Kinase (FAK) and FAK Related Non-Kinase (FRNK) in Leukocyte Recruitment
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AbstractEndothelial cells form the first barrier that leukocytes have to breach in order to reach the site of inflammation. Focal adhesions are the cellular structures that connect the endothelial cytoskeleton to the extracellular matrix. Focal adhesion kinase (FAK) is a signalling protein that is localized to focal adhesions and becomes phosphorylated during leukocyte recruitment. The main aim of this thesis research was to test the hypothesis that FAK is required for leukocyte transmigration. We used in vitro models of TNF-α - and IL-4-mediated inflammation to examine the recruitment and transmigration of neutrophils and eosinophils, respectively. We observed shear-independent loss of FAK in the proximity of neutrophil transmigration. Downregulation of FAK by siRNA, or disruption of FAK signalling by overexpression of FRNK (FAK-related non kinase, an inhibitor of FAK) decreased neutrophil transmigration without interfering with the TNF-α signalling pathway, implicating a functional role for FAK during neutrophil transmigration. In contrast, downregulation of FAK had no effect on eosinophil transmigration. Surprisingly, FRNK overexpression reduced eosinophil transmigration and this was shown to occur independently of FAK. FRNK blocked eosinophil recruitment and transmigration by preventing transcription and protein expression of IL-4 mediated VCAM-1 and CCL26. Interestingly, the FAK inhibitor FAK-II also blocked VCAM-1 and CCL26 expression through an unknown mechanism that we propose involves enhanced availability of the scaffolding domains of inactivated FAK. Finally, we attempted to determine how FRNK regulates expression of VCAM-1. Phosphorylation and nuclear localization of the transcription factor STAT6 were unaffected by FRNK. We found that, in addition to STAT6, IL-4 also regulates expression of GATA6, which induced VCAM-1 expression, and that loss of GATA6 attenuated eosinophil recruitment and transmigration. FRNK reduced IL-4-induced transcription of GATA6 but not its translation. Although we were unable to determine how FRNK regulates VCAM-1, endogenous FRNK was shown to be regulated by IL-4, suggesting an anti-inflammatory role for FRNK in an IL-4 model of leukocyte recruitment.
CitationSharma, R. (2017). The Role of Endothelial Focal Adhesion Kinase (FAK) and FAK Related Non-Kinase (FRNK) in Leukocyte Recruitment (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/28323
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