Hypoxia-inducible factor and nuclear factor kappa-B activation in blood–brain barrier endothelium under hypoxic/reoxygenation stress Ken A. Witt,* Karen S. Mark, Jason Huberà and Thomas P. Davis* *Department of Pharmacology, University of Arizona, College of Medicine, Arizona, USA  Pharmacology Division, College of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri, USA àDepartment of Pharmaceutical Sciences, West Virginia University School of Pharmacy, Morgantown, West Virginia, USA Abstract This investigation focuses on transcription factor involvement in blood–brain barrier (BBB) endothelial cell-induced altera- tions under conditions of hypoxia and post-hypoxia/reoxy- genation (H/R), using established in vivo/ex vivo and in vitro BBB models. Protein/DNA array analyses revealed a corre- lation in key transcription factor activation during hypoxia and H/R, including NFjB and hypoxia-inducible factor (HIF)1. Electrophoretic mobility shift assays confirmed NFjB 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 HIF1a protein expression in both cyto- plasmic and nuclear fractions, in ex vivo and in vitro models. Co-immunoprecipitation of HIF1a and HIF1b 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 NFjB-p65 protein in both cytoplasmic and nuclear fractions. Co-immunoprecipitation of NFjB-p65 with NFjB-p50 was shown in the nuclear fraction under con- ditions 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. Keywords: blood-brain barrier, hypoxia, hypoxia-inducible factor, nuclear factor kappa-B, reoxygenation. J. Neurochem. (2005) 92, 203–214. The blood–brain barrier (BBB) is a physical and metabolic barrier that separates the peripheral circulation from the CNS, and serves to regulate and protect the microenvironment of the brain. Disruption of the BBB occurs in a number of pathological conditions, including inflammatory disorders, Alzheimer’s disease, diabetes, multiple sclerosis and stroke/ reperfusion (Hawkins et al. 1991; Banks et al. 1997; Abbruscato and Davis 1999; Kalaria 1999; Huber et al. 2001). Hypoxia and reoxygenation changes associated with various disease states significantly influence the BBB, with subsequent effects on more sensitive neural tissue. Clarifying the extent and manner of BBB alterations induced by hypoxia and/or post-hypoxic reoxygenation (H/R) remains a considerable task. Regulation and activation of transcription factors (TFs) under such conditions is vital to our under- standing of these BBB changes. Cells have developed elaborate strategies to respond to increased or decreased oxygen tension. Specific cellular systems detect excessive hypoxia and associated reactive oxygen species, leading to the activation of specific TFs and expression of related target genes (D’Angio and Finkelstein 2000). The oxygen and redox-sensitive TFs hypoxia-indu- cible factor (HIF)1 and nuclear factor kappa-B (NFjB) are Received July 27, 2004; revised manuscript received September 10, 2004; accepted September 14, 2004. Address correspondence and reprint requests to Dr Thomas P. Davis, Department of Pharmacology, University of Arizona, College of Medi- cine, PO Box 245050, Tucson, AZ 85724, USA. E-mail: davistp@u.arizona.edu Abbreviations used: AP-1, activator protein 1; BBB, blood–brain barrier; BBMEC, bovine brain microvessel endothelial cell; BSA, bovine serum albumin; EMSA, electrophoretic mobility shift assay; HAS, hypoxia-inducible factor ancillary sequence; HBS, hypoxia-inducible factor binding site; HIF, hypoxia-inducible factor; H/R, hypoxia/reoxy- genation; HRE, hypoxia reponse element; NFjB, nuclear factor kappa- B; PKC, protein kinase C; PI3K, phosphatidylinositol 3-kinase; SDS, sodium dodecyl sulfate; RREB, Ras-responsive transcription element; TF, transcription factor; TJ, tight junction; VEGF, vascular endothelial growth factor; ZO-1, zona occluden-1. Journal of Neurochemistry , 2005, 92, 203–214 doi:10.1111/j.1471-4159.2004.02871.x Ó 2005 International Society for Neurochemistry, J. Neurochem. (2005) 92, 203–214 203