Apurinic apyrimidinic endonuclease redox factor-1 (APE1/Ref-1) is known as multifunctional protein with redox activity. Now APE1/Ref-1 was proved to be secreted from stimulated cells in previous our study. 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.Design and method:
During an inhibitor of histone deacetylase, trichostatin A (TSA)-mediated intracellular acetylation, a time-dependent increase in secreted APE1/Ref-1 was confirmed by an enzyme-linked immunosorbent assay and immunoblotting.Results:
TSA-induced intracellular acetylation in culture significantly suppressed vascular cell adhesion molecule-1 (VCAM-1). Interestingly, secreted APE1/Ref-1 was acetylated and rapidly converted to the nonacetylated, native form base on the removal kinetics of the acetyl moiety. 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, according to biochemical reducing activity and a biotin-switch assay. 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.Conclusions:
APE1/Ref-1 secreted from TNF-α-stimulated endothelial cells in response to intracellular acetylation functions as an endogenous inhibitor of vascular inflammation, leading to the inhibition of TNF-α binding to TNFR1 by reductive conformational change.