Hypoxia-inducible factor and nuclear factor kappa-B activation in blood-brain barrier endothelium under hypoxic/reoxygenation stress.
This investigation focuses on transcription factor involvement in blood-brain barrier (BBB) endothelial cell-induced alterations under conditions of hypoxia and post-hypoxia/reoxygenation (H/R), using established in vivo/ex vivo and in vitro BBB models. Protein/DNA array analyses revealed a correlation in key transcription factor activation during hypoxia and H/R, including NFkappaB and hypoxia-inducible factor (HIF)1. Electrophoretic mobility shift assays confirmed NFkappaB and HIF1 binding activity ex vivo and in vitro, under conditions of hypoxia and H/R. Hypoxia- and H/R-treated BBB endothelium showed increased HIF1alpha protein expression in both cytoplasmic and nuclear fractions, in ex vivo and in vitro models. Co-immunoprecipitation of HIF1alpha and HIF1beta was shown in the nuclear fraction under conditions of hypoxia and H/R in both models. Hypoxia- and H/R-treated BBB endothelium showed increased expression of NFkappaB-p65 protein in both cytoplasmic and nuclear fractions. Co-immunoprecipitation of NFkappaB-p65 with NFkappaB-p50 was shown in the nuclear fraction under conditions of hypoxia and H/R in the ex vivo model, and after H/R in the in vitro model. These data offer novel avenues in which to alter and/or investigate BBB activity across model systems and to further our understanding of upstream regulators during hypoxia and H/R.
Witt
Ken A
KA
Department of Pharmacology, University of Arizona, College of Medicine, Arizona 85724, USA.
Mark
Karen S
KS
Huber
Jason
J
Davis
Thomas P
TP
D000818:Animals; D001812:Blood-Brain Barrier / Q000378:metabolism*; D002417:Cattle; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D005260:Female; D002534:Hypoxia, Brain / Q000235:genetics / Q000378:metabolism*; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013312:Stress, Physiological / Q000235:genetics / Q000378:metabolism; D051996:Transcription Factor RelA; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; C497444:Hif1a protein, rat; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D016328:NF-kappa B; D009687:Nuclear Proteins; D021122:Protein Subunits; D051996:Transcription Factor RelA; D014157:Transcription Factors