Pre-B-cell-colony-enhancing factor is critically involved in thrombin-induced lung endothelial cell barrier dysregulation

Abstract

Prior genomic and genetic studies identified pre-B-cell colony-enhancing factor (PBEF) as a novel candidate gene and biomarker in acute lung injury (ALI). As increased vascular permeability is a cardinal feature of ALI, we assessed the role of PBEF in in vitro vascular barrier regulation using confluent human pulmonary artery endothelial cell (HPAEC) monolayers. Reductions in PBEF protein expression (>70%) by siRNA significantly attenuated EC barrier dysfunction induced by the potent edemagenic agent, thrombin, reflected by reductions in transendothelial electric resistance (TER, ∼60% reduction). Furthermore, PBEF siRNA blunted thrombin-mediated increases in Ca²⁺ entry, polymerized actin formation, and myosin light chain phosphorylation, events critical to the thrombin-mediated permeability response. Finally, PBEF siRNA also significantly inhibited thrombin-stimulated increase of IL-8 secretion in HPAEC, a chemokine known to induce actin fiber formation and intercellular gap formation of endothelial cells. Taken together, these studies demonstrate that PBEF may be required for complete expression of the thrombin-induced inflammatory response and reveal potentially novel role for PBEF in the regulation of EC Ca²⁺-dependent cytoskeletal rearrangement and endothelial barrier dysfunction. Ongoing studies will continue to address the molecular mechanisms by which PBEF contributes to ALI susceptibility.

Introduction

Pre-B-cell-colony-enhancing factor (PBEF), until recently, was a relatively unknown cytokine (less than 10 PubMed citations prior to 2000) despite the isolation of its cDNA from human lymphocytes in 1994 (Samal et al., 1994). More recently, acute mechanical distention was found to upregulate PBEF expression (Nemeth et al., 2000), and multiple inflammatory stimuli (including lipopolysaccharide, interleukin (IL)-1β, tumor necrosis factor (TNF)α, IL-6) all significantly increased in vitro PBEF expression (Ognjanovic et al., 2001). Using cDNA arrays, PBEF was found to be highly expressed in human gestational membranes delivered preterm with chorioamnionitis compared to those without chorioamnionitis, suggesting that PBEF is a novel inflammatory gene implicated in preterm labor (Marvin et al., 2002). Using a suppressive subtractive hybridization in the neutrophil, Jia et al. (2004) detected PBEF as an upregulated gene by IL-1β and suggested that PBEF may play a requisite role in the delayed neutrophil apoptosis of clinical and experimental sepsis. Using the differential display technique, Fukuhara et al. (2005) recently described significantly higher PBEF (in this report termed visfatin) expression in human visceral fat compared to subcutaneous fat and during 3T3-L1 adipocyte differentiation in vitro. Interestingly, PBEF/visfatin exerted both in vitro and in vivo insulin-mimetic actions, indicating a potential role in glucose homeostasis and a potential novel role in metabolic disorders.

Acute lung injury (ALI) is characterized by a diffuse intense inflammatory process and by damage to both endothelial and epithelial cell barriers, resulting in marked extravasation of vascular fluid into the alveolar airspace (Matthay et al., 2003). Extensive microarray-based gene expression profiling in canine, murine, and human ALI models revealed PBEF as a highly expressed gene, results validated by quantitative real time PCR, protein expression, and immunohistochemistry (Ye et al., 2005). Our SNP discovery studies identified 11 single nucleotide polymorphisms (SNPs) in the PBEF gene proximal promoter region (+1 to −3000 bp) with genotyping of 2 SNPs (T-1001G and C-1543T), revealing that the GC haplotype had a 7.7-fold higher risk of ALI (Ye et al., 2005). Taken together, these results strongly indicate PBEF as a potential novel candidate gene and biomarker in ALI.

The molecular underpinning of PBEF in the pathogenesis of ALI, is unknown, however, our results again utilizing animal models of ALI and samples from ALI human patients as well as in vitro cell culture experiments (Ye et al., 2005) strongly indicated PBEF as a dual sensor or transducer for both increased mechanical stress and inflammation. As both events are known key risk factors in the increased pulmonary permeability and the susceptibility to the pathogenesis of ALI, we hypothesize that PBEF overexpression may adversely affect the endothelial barrier function, thus contributing to the increased pulmonary permeability and the pathogenesis of ALI. In this study, we examined whether downregulating PBEF protein expression by the PBEF-specific siRNA would affect thrombin-induced decreases in human lung endothelial cell barrier function (measured by the decreased transendothelial electric resistance or TER), increased cytoskeletal rearrangement, and secretion of the proinflammatory cytokine IL-8. Results from these studies support a potential role for PBEF in the molecular regulation of endothelial barrier function and shed light on novel role of PBEF in the pathogenesis of ALI.