FAK maintenance of endothelial mechanotransduction controls epigenetic repression of KLF2 and S1PR1 transcription

Abstract

Sphingosine‐1‐phosphate (S1P) activation of sphingosine‐1‐phosphate receptor 1 (S1PR1) in endothelial cells (ECs) strengthens the endothelial barrier and prevents lung injury. However mechanisms regulating EC‐S1PR1 expression remains unclear. Recently, we showed that endothelial‐specific deletion of focal adhesion kinase (FAK) in mice severely disrupted lung vascular barrier function. Microarray analysis of FAK depleted cells showed a marked reduction in S1PR1 expression. Thus, we tested the hypothesis that impaired S1PR1 synthesis is responsible for defective endothelial barrier repair in EC‐FAK−/− mice. We confirmed that FAK deletion markedly reduced S1PR1 expression both at the mRNA and protein level in lungs and ECs whereas the expression of S1PR2 and S1PR3 and S1P generating enzymes, sphingosine kinases (SPHK1 and SPHK2) was not reduced. Consistently, FAK depleted ECs and mice conditionally lacking FAK in pulmonary ECs failed to anneal adherens junctions and showed impairment of endothelial barrier function under basal condition and even after S1P application. Rescuing S1PR1 expression in FAK null ECs restored barrier function in the pulmonary vessels of EC‐FAK null mice. Next, we screened for potential transcription factors binding to the S1PR1 promoter which included KLF2, Sox2, FoxP1, GATA1, GATA4 and ERG1. Intriguingly, we observed that rescuing KLF2 expression in FAK depleted ECs restored S1PR1 expression and endothelial barrier function to the levels observed in control cells. In other studies, we observed that KLF2 increased S1PR1 promoter activity while the S1PR1 cDNA lacking KLF2 binding sites failed to increase S1PR1 promoter activity, demonstrating that KLF2 can promote endothelial barrier function in the absence of EC‐FAK by transducing S1PR1. Because FAK maintains intracellular tension by balancing the activities of RhoA and Rac1 GTPases and KLF2 mRNA expression is regulated by methylation of CpG islands, we next address the possible role of increased cellular tension and DNA methyltransferases (DNMT) in suppressing KLF2 gene transcription and thereby S1PR1 expression in EC‐FAK null mice. We found that DNMT activity was elevated by 3‐fold in FAK depleted ECs in a RhoA‐dependent manner. Intriguingly, we found that depletion of FAK increased EC‐stiffness, which was associated with hypermethylation of KLF2 by DNMT3a. Interestingly, inhibition of RhoA in FAK depleted cells restored KLF2 and S1PR1 expression. Thus, our data describes the novel role of FAK in suppressing cellular tension driven‐epigenetic modification of KLF2 and S1PR1 and thereby vascular integrity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.