Optimal expression of endothelial-enriched tunica interna endothelial cell kinase (Tie2) and vascular endothelial cadherin (VE-cad) in endothelial cells (ECs) is required to form restrictive endothelial barrier and to maintain vascular homeostasis. Acute lung injury (ALI) is a complex inflammatory disease associated with increased lung vascular permeability. Rapid reduction in the expression of Tie2 and VE-cad in ECs contributes to ALI. Studies proposed in this application will test the central hypothesis that the calcium/calmodulin (Ca2+/CaM)-dependent kinase CaMKK?-mediated expression of the transcription factor Elf2 promotes the resolution of inflammatory lung injury through the expression of Tie2 and VE-cad in ECs. This project was inspired by our seminal observations that Camkk? deficient (Camkk??/?) mice are unusually susceptible to LPS-induced lung injury and that expression of both CaMKK? and Elf2 is downregulated in lung endothelia from septic patients. We discovered that CaMKK? signaling downstream of TLR4 and PAR-1 (a GPCR) mediates EC expression of Elf2, which in turn induces EC-specific transcription of the receptor tyrosine kinase Tie2, and VE-cad. In Camkk??/? mice, the DNA methyltransferase inhibitor 5-azacytidine or expression of wild type (WT) but not kinase-defective CaMKK?, restored the expression of Tie2 and VE-cad. Genome-wide methylation analysis showed that the gene encoding the transcription factor Elf2 was hyper-methylated in ECs of Camkk??/? mice. Further, methyl- CpG-binding protein 2 (MeCP2), which binds methylated-CpG and thereby represses transcription, was associated with regulatory regions of the Elf2 gene in Camkk??/? mice. Consistent with these findings, Elf2 expression was markedly reduced in ECs of Camkk??/? mice. Interestingly, EC-specific deletion of either DNA methyltransferase Dnmt3b (Dnmt3bEC?/?) or Mecp2 (Mecp2EC?/?) in mice, augmented Elf2 expression in ECs. Importantly, in EC-specific Elf2 knockout (Elf2EC?/?) mice, expression of Tie2 and VE-cad was dramatically reduced. Based on these novel findings, in Aim 1a, we will test the hypothesis that DNA methyl transferase DNMT3b mediates methylation of Elf2-gene promoter in quiescent ECs and in Aim 1b, we will test the hypothesis that the methyl CpG binding protein MeCP2, binds methylated-CpG in the promoter regions of the Elf2 gene and inactivates Elf2 transcription.
In Aim 2, we will test the hypothesis that CaMKK? activated downstream of TLR4 and/or PAR-1 mediates phosphorylation of MeCP2 residue S421, which in turn induces the expression of Elf2 in ECs.
In Aim 3, we will test the hypothesis that Elf2 activation is required for the optimal expression of Tie2 and VE-cad in ECs and thus repair of the lung endothelial barrier. We will employ biochemical, molecular, and imaging approaches to define the underlying mechanisms. Importantly, we will use EC-restricted knockout mouse models (Dnmt3bEC?/?, Mecp2EC?/?, Camkk?EC?/? and Elf2EC?/?) created by us to accomplish the goals. Our hope is that these studies will identify therapeutic targets to reverse sepsis-induced acute lung injury based on a deeper understanding of endogenous EC repair programs.
Acute lung injury (ALI) has high mortality because of edema resulting from pulmonary vascular endothelium dysfunction and currently there is no specific treatment for this disease. Healthy endothelial cells prevent edema formation through expression of receptor tyrosine kinase Tie2 and vascular endothelial (VE)-cadherin (VE-cad) to maintain blood vessel barrier integrity. The present investigation will determine how expression of the transcription factor Elf2 in endothelial cells induces the expression of Tie2 and VE-cad needed to repair the injured blood vessel endothelial barrier.
