Abstract
Apurinic apyrimidinic endonuclease 1/Redox factor-1 (APE1/Ref-1) is a multifunctional protein with redox activity and is proved to be secreted from stimulated cells. The aim of this study was to evaluate the functions of extracellular APE1/Ref-1 with respect to leading anti-inflammatory signaling in TNF-α-stimulated endothelial cells in response to acetylation. Treatment of TNF-α-stimulated endothelial cells with an inhibitor of deacetylase that causes intracellular acetylation, considerably suppressed vascular cell adhesion molecule-1 (VCAM-1). During TSA-mediated acetylation in culture, a time-dependent increase in secreted APE1/Ref-1 was confirmed. The acetyl moiety of acetylated-APE1/Ref-1 was rapidly removed based on the removal kinetics. Additionally, recombinant human (rh) APE1/Ref-1 with reducing activity induced a conformational change in rh TNF-α receptor 1 (TNFR1) by thiol-disulfide exchange. Following treatment with the neutralizing anti-APE1/Ref-1 antibody, inflammatory signals via the binding of TNF-α to TNFR1 were remarkably recovered, leading to up-regulation of reactive oxygen species generation and VCAM-1, in accordance with the activation of p66shc and p38 MAPK. These results strongly indicate that anti-inflammatory effects in TNF-α-stimulated endothelial cells by acetylation are tightly linked to secreted APE1/Ref-1, which inhibits TNF-α binding to TNFR1 by reductive conformational change, with suggestion as an endogenous inhibitor of vascular inflammation.
Highlights
Chronic vascular inflammation plays a key role in the pathogenesis of atherosclerosis and other vascular disease[1]
The histone deacetylase inhibitor (HDACi), trichostatin A (TSA) inhibits the expression of the cell adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor (TNF)-α -stimulated endothelial cells[22], but the sequence of events leading to anti-inflammatory effects in the vascular system is still unclear
These results indicated that VCAM-1 expression in TNF-α stimulated endothelial cells is regulated by acetylation/deacetylation, implying a functional role of the APE1/ Ref-1 protein
Summary
Chronic vascular inflammation plays a key role in the pathogenesis of atherosclerosis and other vascular disease[1]. Because the level of secreted APE1/Ref-1 is substantially increased in response to acetylation, we hypothesized that secreted APE1/Ref-1 could be an effective regulator in inflammatory reactions via its reduction. We tested this hypothesis using TNF-α -treated human umbilical vein endothelial cells (HUVECs) as a vascular inflammation model. We provide compelling experimental evidence to indicate that extracellular secreted APE1/Ref-1 in response to intracellular acetylation inhibits inflammatory signaling via a reduction in TNFR1, showing that treatment of anti-APE1/Ref-1 antibody in histone deacetylase inhibitor (HDACi), trichostatin A (TSA)-mediated modulation against TNF-α -stimulated endothelial activation recovers upregulation of adhesion molecule and the generation of ROS
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