Abstract 4913: Glutathione Peroxidase-1 Modulates the Effects of Tumor Necrosis Factor-alpha in Endothelial Cells by Regulating Dual Specificity Phosphatase 4
Reactive oxygen species (ROS) have been implicated in pro-inflammatory signal transduction activated by tumor necrosis factor-α (TNF-α). For this reason, we examined the effect of glutathione peroxidase-1 (GPx-1), an important antioxidant enzyme, on TNF-α-induced changes in gene expression in endothelial cells. In human microvascular endothelial cells (HMVEC), we showed that suppression of GPx-1 mRNA by 96.6% (p<0.0001) with siRNA (siGPx-1) increases ICAM-1 mRNA (3.6-fold, p<0.01) and protein (7.1-fold, p = 0.0002) expression compared to siControl cells, and GPx-1 deficiency augments TNF-α-induced ICAM-1 mRNA (2.1-fold, p<0.0001) and protein (2.5-fold, p = 0.001) expression, in part, by enhancing ROS production and prolonging IκBα degradation and phospho-ERK1/2 activation. The antioxidants, N-acetyl-L-cysteine and diphenyleneiodonium chloride, or adenoviral over-expression of GPx-1 significantly attenuated these TNF-α-mediated responses. Similarly, endothelial cells cultured from GPx-1 knockout mice had augmented TNF-α-mediated ICAM-1 expression, whereas cells from transgenic mice overexpressing GPx-1 showed blunted responses to TNF-α, confirming that GPx-1 modulates the inflammatory response to TNF-α. To analyze further signaling pathways, we performed microarray analysis of HMVEC treated with TNF-α in the presence and absence of GPx-1. Among the genes whose expression changed significantly, dual specificity phosphatase 4 (DUSP4), an antagonist of MAPK signaling, was downregulated 3.2-fold (p<0.005) by GPx-1 suppression. Quantitative real time PCR confirmed that GPx-1 deficiency enhanced DUSP4 suppression by TNF-α (p<0.01). Targeted knockdown of DUSP4 mRNA by 78.5% (p<0.0001) significantly increased TNF-α-mediated ICAM-1 and VCAM-1 gene expression (p<0.01 and p<0.012, respectively), suggesting that loss of DUSP4 is sufficient to augment TNF-α-mediated induction of adhesion molecule expression. Taken together, these data show that GPx-1 modulates endothelial cell activation by TNF-α, in part, by modulating DUSP4 expression. These results highlight a novel mechanism by which GPx-1 modulates ROS-dependent signaling in endothelial cells.