The Role of Endothelial Focal Adhesion Kinase (FAK) and FAK Related Non-Kinase (FRNK) in Leukocyte Recruitment
Abstract
Endothelial 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.
Description
Keywords
Education--Sciences
Citation
Sharma, R. (2017). The Role of Endothelial Focal Adhesion Kinase (FAK) and FAK Related Non-Kinase (FRNK) in Leukocyte Recruitment (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/28323