We hypothesized that a redox-sensitive transcription factor, redox factor-1 (Ref-1) (HAP1, APE, and APEX), was critical in the regulation of endothelial cell survival in response to hypoxia and cytokines, including tumor necrosis factor (TNF)-α. Hypoxia resulted in a significant decrease in Ref-1 protein expression in both human umbilical vein endothelial cells and calf pulmonary artery endothelial cells. The hypoxia-induced decrease in Ref-1 expression was followed by a significant induction of apoptosis as measured by caspase 3 activity and nuclear morphology. Transient upregulation of Ref-1 significantly inhibited hypoxia-induced apoptosis. However, deletion of the redox-sensitive domain of Ref-1 abolished the antiapoptotic effect. We postulated that the antiapoptotic effects of Ref-1 were mediated through nuclear factor-κB (NF-κB). However, blockade of NF-κB with a dominant-negative IκB (S32A/S36A) expression vector had no effect on Ref-1-mediated survival under hypoxic conditions. The second aim of this study was to test the cytoprotective ability of Ref-1 upregulation in response to TNF-induced apoptosis. Ref-1 inhibition of TNF-induced death was associated with a significant potentiation of NF-κB activity. Deletion of the redox-sensitive domain of Ref-1 significantly inhibited TNF-induced NF-κB activation. Moreover, loss of the redox-sensitive domain also abolished the antiapoptotic effect of Ref-1 in response to TNF. To test whether Ref-1 induced activation of NF-κB was necessary to promote survival, we blocked NF-κB activity with a dominant-negative IκB (S32A/S36A). Indeed, blockade of NF-κB activity abolished the ability of Ref-1 to rescue TNF-induced apoptosis. In conclusion, upregulation of Ref-1 promotes endothelial cell survival in response to hypoxia and TNF through NF-κB-independent and NF-κB-dependent signaling cascades, respectively. Moreover, it seems that Ref-1 may act as a critical cofactor, mediating the TNF-induced NF-κB response in the vascular endothelium.
- Nuclear factor-κB