Overexpression of Ref-1 inhibits hypoxia and tumor necrosis factor-induced endothelial cell apoptosis through nuclear factor-kappab-independent and -dependent pathways.
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)-alpha. 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-kappaB (NF-kappaB). However, blockade of NF-kappaB with a dominant-negative IkappaB (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-kappaB activity. Deletion of the redox-sensitive domain of Ref-1 significantly inhibited TNF-induced NF-kappaB 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-kappaB was necessary to promote survival, we blocked NF-kappaB activity with a dominant-negative IkappaB (S32A/S36A). Indeed, blockade of NF-kappaB 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-kappaB-independent and NF-kappaB-dependent signaling cascades, respectively. Moreover, it seems that Ref-1 may act as a critical cofactor, mediating the TNF-induced NF-kappaB response in the vascular endothelium.
Hall
J L
JL
Division of Cardiovascular Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Mass, USA. hallx@tc.umn.edu
Wang
X
X
Van Adamson
Zhao
Y
Y
Gibbons
G H
GH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D019757:Carbon-Oxygen Lyases / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D015687:Cell Hypoxia / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors; D014158:Transcription, Genetic / Q000502:physiology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects
D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016328:NF-kappa B; D009687:Nuclear Proteins; D018124:Receptors, Tumor Necrosis Factor; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D014409:Tumor Necrosis Factor-alpha; D016159:Tumor Suppressor Protein p53; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D019757:Carbon-Oxygen Lyases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase