Inflammation Modulates RLIP76/RALBP1 Electrophile-Glutathione Conjugate Transporter and Housekeeping Genes in Human Blood-Brain Barrier Endothelial Cells

PLoS One. 2015 Sep 25;10(9):e0139101. doi: 10.1371/journal.pone.0139101. eCollection 2015.

Abstract

Endothelial cells are often present at inflammation sites. This is the case of endothelial cells of the blood-brain barrier (BBB) of patients afflicted with neurodegenerative disorders such as Alzheimer's, Parkinson's, or multiple sclerosis, as well as in cases of bacterial meningitis, trauma, or tumor-associated ischemia. Inflammation is a known modulator of gene expression through the activation of transcription factors, mostly NF-κB. RLIP76 (a.k.a. RALBP1), an ATP-dependent transporter of electrophile-glutathione conjugates, modulates BBB permeability through the regulation of tight junction function, cell adhesion, and exocytosis. Genes and pathways regulated by RLIP76 are transcriptional targets of tumor necrosis factor alpha (TNF-α) pro-inflammatory molecule, suggesting that RLIP76 may also be an inflammation target. To assess the effects of TNF-α on RLIP76, we faced the problem of choosing reference genes impervious to TNF-α. Since such genes were not known in human BBB endothelial cells, we subjected these to TNF-α, and measured by quantitative RT-PCR the expression of housekeeping genes commonly used as reference genes. We find most to be modulated, and analysis of several inflammation datasets as well as a metaanalysis of more than 5000 human tissue samples encompassing more than 300 cell types and diseases show that no single housekeeping gene may be used as a reference gene. Using three different algorithms, however, we uncovered a reference geneset impervious to TNF-α, and show for the first time that RLIP76 expression is induced by TNF-α and follows the induction kinetics of inflammation markers, suggesting that inflammation can influence RLIP76 expression at the BBB. We also show that MRP1 (a.k.a. ABCC1), another electrophile-glutathione transporter, is not modulated in the same cells and conditions, indicating that RLIP76 regulation by TNF-α is not a general property of glutathione transporters. The reference geneset uncovered herein should aid in future gene expression studies in inflammatory conditions of the BBB.

MeSH terms

  • ATP-Binding Cassette Transporters / genetics
  • ATP-Binding Cassette Transporters / metabolism
  • Animals
  • Binding Sites
  • Biomarkers / metabolism
  • Blood-Brain Barrier / pathology*
  • Endothelial Cells / drug effects
  • Endothelial Cells / metabolism*
  • Endothelial Cells / pathology
  • GTPase-Activating Proteins / genetics
  • GTPase-Activating Proteins / metabolism
  • Gene Expression Regulation / drug effects
  • Genes, Essential*
  • Genetic Association Studies
  • Glutathione / metabolism*
  • Humans
  • Inflammation / genetics*
  • Inflammation / pathology
  • Mice
  • NF-kappa B / metabolism
  • Promoter Regions, Genetic / genetics
  • Rats
  • Software
  • Tumor Necrosis Factor-alpha / pharmacology

Substances

  • ATP-Binding Cassette Transporters
  • Biomarkers
  • GTPase-Activating Proteins
  • NF-kappa B
  • RALBP1 protein, human
  • Tumor Necrosis Factor-alpha
  • Glutathione

Grant support

The authors have no support or funding to report.