Abstract 13633: TNF-α Activation of NFκB Promotes Human Endothelial Cell Survival by Down-Regulating the Expression of DUSP4, a Dual-Specificity Phosphatase
TNF-α contributes to upregulation of proinflammatory pathways; however, TNF-α may also mediate some cytoprotective pathways. We found that 10 ng/ml TNF-α causes a gradual and sustained downregulation of DUSP4 expression (ANOVA, p<0.0001, pairwise posthoc p<0.05 from 1 hr to 24 hrs compared to untreated) in human microvascular endothelial cells. We hypothesized that suppression of DUSP4, a dual-specificity phosphatase that regulates ERK1/2 activation, will enhance cell survival by increasing sensitivity to growth factor stimulation. In support of this concept, overexpression of DUSP4 with an adenovirus construct (AdDUSP) reduced growth factor mediated-ERK1/2 activation compared to a control adenovirus (AdCtrl). Overexpression of DUSP4 significantly decreased cell number by 42 ± 7 % (± std dev) by direct cell counting, 45 ± 15 % by metabolic dehydrogenase activity assays or 50 ± 12 % by protease-based viability assays and increased the relative proportion of dead cells 2.3 ± 0.2-fold compared to AdCtrl or no virus (ANOVA p<0.01, p<0.05, pairwise). To determine the mechanism by which TNF-α suppresses DUSP4 expression, endothelial cells were pretreated with PDTC, an antioxidant with NFkB inhibitor properties. PDTC pretreatment (50 uM) significantly blocked TNF-α-mediated inflammatory effects, as it inhibited ICAM-1 and VCAM-1 upregulation. This anti-inflammatory treatment also significantly lessened TNF-α-mediated suppression of DUSP4 gene expression from 64 + 5 % to 19% ± 6 % (p<0.005, t-test). To confirm a role for NFκB in DUSP4 suppression, endothelial cells were treated with a dominant negative IkBα adenovirus (AdIkBαDN) prior to TNF-α exposure. AdIkBαDN inhibited TNF-α-mediated migration of p65 to the nucleus, attenuated TNF-α-induced upregulation of adhesion molecule gene expression, and significantly lessened TNF-α-induced DUSP4 suppression from 77 ± 1 % with AdCtrl virus to 12 ± 4 % (ANOVA, P<0.001, pairwise p<0.05). Taken together, these findings illustrate a new pathway by which TNF-α mediated NFκB-activation regulates MAPK-signaling in the endothelium. These pathways may contribute to the upregulation of crucial endothelial cell survival mechanisms following TNF-α exposure, as overexpression of DUSP4 decreases cell growth.
- © 2011 by American Heart Association, Inc.