Focal Adhesion Kinase, Maintenance of Sphingosine 1 Phosphate Receptor 1 Expression and Barrier Function

Formation of leaky blood vessels remains a persistent pathology in several diseases including acute lung injury. Sphingosine-1-phosphate (S1P) is a lipid mediator well known for its ability to induce endothelial barrier function. Formed through metabolism of sphingomyelin, S1P regulates endothelial barrier function by binding to S1P receptor 1 (previously known as endothelial differentiation gene (Edg1). However, mechanisms upregulating S1PR1 expression enabling endothelial barrier formation remains unclear. Recently we showed that loss of focal adhesion kinase (FAK), a non-receptor tyrosine kinase, in endothelial cells spontaneously induced vascular leak. We therefore tested the hypothesis that FAK stabilize vascular barrier function by regulating S1PR1 function. We showed that FAK depletion significantly reduced the expression of S1PR1 at mRNA and protein level. Consistently, FAK depleted ECs and mice conditionally lacking FAK in pulmonary ECs failed to anneal adherens junctions and showed loss endothelial barrier function under basal conditions and even after S1P application. Rescuing S1PR1 expression in FAK null ECs restored barrier function in the pulmonary vessels of EC-FAK-/- mice. Promoter bashing of S1PR1 identified KLF2 (Krϋppel-like transcription factor 2) as the transcription factor regulating S1PR1 transcription downstream of FAK. Hence, rescuing KLF2 in FAK depleted cells rescued S1PR1 expression and S1P response. MEF2 by binding to KLF2 promoter regulates KLF2 expression. Both KLF2 and MEF2 are regulated by mechanical tension. Moreover, KLF2 promoter contains many CpG dinucleotides clusters that can be methylated through activation of DNA methyltransfereases. FAK maintains intracellular ECs tension by suppressing the activity of small GTPase RhoA. Thus, to address the upstream mechanism by which FAK induced KLF2 and thereby S1PR1 we therefore determined if loss of FAK increased ECs tension which then epigenetically modified KLF2 promoter through DNA methylation. Interestingly, we found that depletion of FAK increased cellular stiffness which was associated with hyper-methylation of KLF2. Inhibition of RhoA in FAK depleted cells restored KLF2 and S1PR1 expression. Our data for the first time describe FAK regulation of cellular tension as the cause of epigenetic modification of KLF2 and S1PR1 signaling which is critical for forming stable endothelial barrier.