A20 Blocks Endothelial Cell Activation through a NF-κB-dependent Mechanism

Abstract

The A20 gene product is a novel zinc finger protein originally described as a tumor necrosis factor α (TNF)-inducible early response gene in human umbilical vein endothelial cells (HUVEC). Its described function is to block TNF-induced apoptosis in fibroblasts and B lymphocytes, but more recently it has also been shown to play a role in lymphoid cell maturation. The mechanism of action of A20 is unknown. The aim of our study was to assess the effect of A20 upon endothelial cell activation. By transfecting bovine aortic endothelial cells (BAEC) with A20 as well as reporter constructs consisting of the promoters of genes known to be up-regulated during endothelial cell activation, i.e. E-selectin, interleukin (IL)-8, tissue factor (TF), and inhibitor of nuclear factor κBα (IκBα), we demonstrate that A20 expression inhibits gene up-regulation associated with TNF, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), and hydrogen peroxide (H2O2)-induced endothelial cell (EC) activation. The mechanism of action of A20 is in part, or totally, due to the blockade of nuclear factor κB (NF-κB), as shown by its ability to suppress the activity of a NF-κB reporter. This effect is specific, as A20 does not block a noninducible, constitutively expressed reporter, Rous sarcoma virus-luciferase (RSV-LUC); nor does it block the c-Tat-inducible, NF-κB-independent reporter, human immunodeficiency virus-chloramphenicol acetyltransferase (HIV-CAT). How A20 blocks NF-κB is unclear, although we demonstrate that it does not affect p65 (RelA)-mediated gene transactivation. The inhibition of endothelial cell activation by A20 is a novel function for A20. The A20 gene product is a novel zinc finger protein originally described as a tumor necrosis factor α (TNF)-inducible early response gene in human umbilical vein endothelial cells (HUVEC). Its described function is to block TNF-induced apoptosis in fibroblasts and B lymphocytes, but more recently it has also been shown to play a role in lymphoid cell maturation. The mechanism of action of A20 is unknown. The aim of our study was to assess the effect of A20 upon endothelial cell activation. By transfecting bovine aortic endothelial cells (BAEC) with A20 as well as reporter constructs consisting of the promoters of genes known to be up-regulated during endothelial cell activation, i.e. E-selectin, interleukin (IL)-8, tissue factor (TF), and inhibitor of nuclear factor κBα (IκBα), we demonstrate that A20 expression inhibits gene up-regulation associated with TNF, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), and hydrogen peroxide (H2O2)-induced endothelial cell (EC) activation. The mechanism of action of A20 is in part, or totally, due to the blockade of nuclear factor κB (NF-κB), as shown by its ability to suppress the activity of a NF-κB reporter. This effect is specific, as A20 does not block a noninducible, constitutively expressed reporter, Rous sarcoma virus-luciferase (RSV-LUC); nor does it block the c-Tat-inducible, NF-κB-independent reporter, human immunodeficiency virus-chloramphenicol acetyltransferase (HIV-CAT). How A20 blocks NF-κB is unclear, although we demonstrate that it does not affect p65 (RelA)-mediated gene transactivation. The inhibition of endothelial cell activation by A20 is a novel function for A20.