CaMKKβ regulates transcription factor Elf2 gene methylation to maintain endothelial junctional barrier integrity

The expression of endothelial-enriched receptor tyrosine kinase Tie2 and vascular endothelial cadherin VE-cadherin in endothelial cells (EC) is essential for maintaining endothelial barrier integrity and vascular homeostasis. The Ets family of transcription factors plays a critical role in regulating the expression of Tie2 and VE-cadherin in EC. However, the transcriptional regulation of these factors remains poorly understood. In this study, we performed whole genome methylation analysis and found that the gene encoding the Ets family transcription factor Elf2/Nerf2 was hypermethylated in Camkkβ-deficient ( Camkkβ ^-/- ) mice. Notably, these mice exhibited significantly reduced mRNA and protein levels of Elf2, Tie2, and VE-cadherin, along with uncontrolled lung vascular injury following LPS challenge. Additionally, either depletion of CaMKKβ in human lung microvascular endothelial cells or EC-specific deletion of Camkkβ in mice led to a decrease in the expression of Elf2, Tie2, and VE-cadherin. Furthermore, administration of the DNA methyltransferase inhibitor 5-azacytidine (5-AZA) or EC-specific expression of wild-type CaMKKβ but not the kinase-defective mutant restored the expression of Elf2, Tie2, and VE-cadherin in Camkkβ ^-/- mice. We find that under baseline conditions, methyl CpG binding protein MeCP2 repressed Elf2 expression in EC. Importantly, in EC-restricted Elf2 knockout mice ( Elf2 ^1EC ), lung endothelial barrier integrity was compromised due to dramatically reduced levels of Tie2 and VE-cadherin. Together, these findings underscore the crucial role of CaMKKβ in regulating endothelial junctional barrier integrity by controlling Elf2 expression.