title: Nrf2 signaling increases expression of ATP-binding cassette subfamily C mRNA transcripts at the blood-brain barrier following hypoxia-reoxygenation stress.
abstract: BACKGROUND
  Strategies to maintain BBB integrity in diseases with a hypoxia/reoxygenation (H/R) component involve preventing glutathione (GSH) loss from endothelial cells. GSH efflux transporters include multidrug resistance proteins (Mrps). Therefore, characterization of Mrp regulation at the BBB during H/R is required to advance these transporters as therapeutic targets. Our goal was to investigate, in vivo, regulation of Abcc1, Abcc2, and Abcc4 mRNA expression (i.e., genes encoding Mrp isoforms that transport GSH) by nuclear factor E2-related factor (Nrf2) using a well-established H/R model.
  
  
  METHODS
  Female Sprague-Dawley rats (200-250 g) were subjected to normoxia (Nx, 21% O2, 60 min), hypoxia (Hx, 6% O2, 60 min) or H/R (6% O2, 60 min followed by 21% O2, 10 min, 30 min, or 1 h) or were treated with the Nrf2 activator sulforaphane (25 mg/kg, i.p.) for 3 h. Abcc mRNA expression in brain microvessels was determined using quantitative real-time PCR. Nrf2 signaling activation was examined using an electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) respectively. Data were expressed as mean ± SD and analyzed via ANOVA followed by the post hoc Bonferroni t test.
  
  
  RESULTS
  We observed increased microvascular expression of Abcc1, Abcc2, and Abcc4 mRNA following H/R treatment with reoxygenation times of 10 min, 30 min, and 1 h and in animals treated with sulforaphane. Using a biotinylated Nrf2 probe, we observed an upward band shift in brain microvessels isolated from H/R animals or animals administered sulforaphane. ChIP studies showed increased Nrf2 binding to antioxidant response elements on Abcc1, Abcc2, and Abcc4 promoters following H/R or sulforaphane treatment, suggesting a role for Nrf2 signaling in Abcc gene regulation.
  
  
  CONCLUSIONS
  Our data show increased Abcc1, Abcc2, and Abcc4 mRNA expression at the BBB in response to H/R stress and that Abcc gene expression is regulated by Nrf2 signaling. Since these Mrp isoforms transport GSH, these results may point to endogenous transporters that can be targeted for BBB protection during H/R stress. Experiments are ongoing to examine functional implications of Nrf2-mediated increases in Abcc transcript expression. Such studies will determine utility of targeting Mrp isoforms for BBB protection in diseases with an H/R component.
authors.affiliation: Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, 1295 N. Martin Avenue, P.O. Box 210202, Tucson, 85721, AZ, USA.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Ibbotson
authors.affiliation: Department of Pharmacology, College of Medicine, University of Arizona, 1501 N. Campbell Avenue, P.O. Box 245050, Tucson, AZ, 85724-5050, USA.
authors.forename: Joshua
authors.initials: J
authors.lastname: Yell
authors.affiliation: Department of Pharmacology, College of Medicine, University of Arizona, 1501 N. Campbell Avenue, P.O. Box 245050, Tucson, AZ, 85724-5050, USA. pronald@email.arizona.edu.
authors.forename: Patrick T
authors.initials: PT
authors.lastname: Ronaldson
mesh_terms: D000818:Animals; D016588:Anticarcinogenic Agents / Q000494:pharmacology; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000378:metabolism*; D001921:Brain / Q000166:cytology; D047369:Chromatin Immunoprecipitation; D004195:Disease Models, Animal; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D006931:Hyperbaric Oxygenation; D000860:Hypoxia / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D017879:Isothiocyanates / Q000494:pharmacology; D055806:Microvessels / Q000187:drug effects / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism*; D051267:NF-E2-Related Factor 2 / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013454:Sulfoxides; D013997:Time Factors
chemical_list: D016588:Anticarcinogenic Agents; D017879:Isothiocyanates; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D051267:NF-E2-Related Factor 2; D012333:RNA, Messenger; D013454:Sulfoxides; C495637:Nfe2l2 protein, rat; C016766:sulforaphane
keywords: Blood–brain barrier; Endothelial cell; Hypoxia; Multidrug resistance proteins; Nrf2 signaling; Transporters