Pre-B-cell colony enhancing factor (PBEF) is a unique, little-studied cytokine, whose relevance to the lung pathophysiology is unknown. Recent work by the PI has provided the first evidence of PBEF expression in lung tissues and overexpression in acute lung injury (ALI) as well as the association of PBEF genetic variants with susceptibility to ALI. The haplotype weighted analysis of single nucleotide polymorphism (SNP) T-1001G and C-1543T in the PBEF gene promoter revealed a susceptible haplotype GC with a 7.7-fold higher risk of ALI. Reporter gene assays indicated that theses variants affected transcription rate. Our recent data suggest that PBEF affects pulmonary endothelial cell permeability in vitro and increased pulmonary edema in C57 BL/6 mice in vivo. In addition, stealth PBEF siRNA significantly inhibited TNF-alpha mediated increase of IL-8 secretion, a known edematogenic factor in ALI, in pulmonary cells. All these results support PBEF as a potential novel candidate gene and biomarker in ALI. To further detail underlying molecular mechanisms of PBEF in the pathogenesis of ALI, we hypothesize that patients with the genetically- determined susceptible haplotype in the PBEF promoter region are more susceptible to the upregulation by mechanical forces and inflammatory stimuli, two most inciting risk factors to ALI;increased PBEF expression contribute to the increased susceptibility to ALI by stimulating expression of other inflammatory cytokines such as IL-8, which leads to the pulmonary artery endothelial and epithelial cell barrier dysfunction and increased pulmonary permeability, resulting in the pathogenesis of ALI. The goal of this application is to define the physiologic and molecular significance of the identified PBEF polymorphisms. Furthermore, we will determine the pathophysiological role of PBEF in inflammatory lung injury with a specific focus on a role of PBEF in increased lung vascular leak/permeability, a pathophysiological feature of ALI, both in tissue culture system in vitro and in Pbef gene knock out C57 BL/6 mice in vivo.
Specific Aim 1 will functionally characterize PBEF gene promoter SNPs utilizing the reporter gene and electrophoretic mobility shift assays.
Specific Aim 2 will examine the role of PBEF gene expression in endothelial cell barrier regulation and contractility in vitro by evaluating endothelial cell transendothelial electric resistance, stress fiber formation, and phosphorylation of myosin light chains.
Specific Aim 3 will define the signal transduction pathways of PBEF-mediated endothelial barrier regulation by examining PBEF- regulated genes and interacting partners.
Specific Aim 4 will examine the in vivo role of PBEF in a murine model of ALI using Pbef knock out and over-expressing animals. Together, these novel and highly translational studies using the genomic and genetic approaches will provide new insight into molecular mechanisms of PBEF function in ALI, which may lead to the development of novel diagnostic modalities and therapeutic strategies in ALI.
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