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

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

TNF-α induces the expression of inflammation markers in blood-brain barrier endothelial cells.

<p>Expression levels of <i>ICAM1</i> (Panel A), <i>VCAM1</i> (Panel B) and <i>MCP1</i> (Panel C) were monitored by qRT-PCR prior (control) and 2 to 48 hours following TNF-α treatment. Expression levels were normalized against <i>PSMB6</i> / <i>HPRT1</i> as a reference geneset (Normalization Factor; black graphs), or against single genes, here <i>GAPDH</i> (grey graphs) or <i>B2M</i> (white graphs). Results are presented as arithmetic means ± standard deviations. Statistical significance: * signifies p < 0.05, ** p < 0.01, and *** p < 0.001; lack of asterisks denotes lack of a statistically significant difference.</p

Gene expression variability of the PSMB6, HPRT1, B2M and GAPDH genes in human cells and diseases.

<p>Gene expression of PSMB6 (<b>A</b>), HPRT1 (B), GAPDH (C), and B2M (D) was assessed across 5372 human tissue samples representing 369 tissues and cell types or diseases clustered into 15 meta-groups (indicated on top of figure). Red and blue arrows indicate expression significantly above (Up) or below (Down) median expression across 15 meta-groups, respectively. Medians of t-statistics and associated p-values are indicated. Ordinate axis: Log2 expression.</p

Candidate reference genes ranked according to <i>geNorm</i>.

<p>M values were calculated as average expression stability of reference genes during stepwise exclusion of the least stable control. V values were calculated as the pairwise variation between two sequential Normalization Factors.</p

Candidate reference genes ranked according to <i>NormFinder</i>.

<p>In this analysis, genes ranked 1–4 were deemed highly stable, whereas genes ranked 8–9 were deemed highly unstable.</p

Modulation of gene expression by drugs or disease state across several tissue and cell types.

<p>Expression of 13 previously reported 'universal reference genes' and of the most and least stably expressed genes uncovered in this study was monitored across 5372 human samples representing 369 different cell and tissue types, disease states and cell lines. Data show the number of studies documenting gene expression modulation for the considered parameters.</p

TNF-α induces <i>RLIP76</i> but not <i>MRP1</i> gene expression.

<p>Expression levels of <i>RLIP76</i> (Panel A) and <i>MRP1</i> (Panel B) were monitored by qRT-PCR prior and after TNF-α treatment for 2–48 hours relative to the <i>PSMB6/HPRT1</i> reference geneset (blue bars), <i>GAPDH</i> (red bars), or to <i>B2M</i> (brown bars). Results are presented as arithmetic means ± standard deviations. Statistical significance: * signifies p < 0.05, *** signifies p < 0.001; lack of asterisks denotes lack of a statistically significant difference.</p

Comparative analysis of human, murine and rat <i>RLIP76</i> promoter sequences unveils conserved NF-κB binding sites.

<p>Human (<i>Homo sapiens</i>: <i>H</i>.<i>s</i>), murine (<i>Mus musculus</i>: <i>M</i>.<i>m</i>) and rat (<i>Rattus norvegicus</i>: <i>R</i>.<i>n</i>) DNA sequences spanning -2 to +0.5 kb relative to the transcription start site (+0 kb) were retrieved using the Cold Spring Harbor Laboratory promoter database (<a href="http://rulai.cshl.edu/cgi-bin/CSHLmpd2/promExtract.pl?" target="_blank">http://rulai.cshl.edu/cgi-bin/CSHLmpd2/promExtract.pl?</a>). Sequence alignments were performed using NCBI Blast platform (<a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi" target="_blank">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a>), and GGGRNNYYCC (whereby R is a purine and Y is a pyrimidine) was used as the consensus NF-κB binding site. Orientation of the binding sites is shown with arrow heads, with read arrowheads corresponding to evolutionarily conserved and black arrowheads non-conserved binding sites.</p

Statistical output of the <i>Bestkeeper</i> analysis.

<p>Candidate reference genes are listed according to increasing standard deviations (SD). Consistency between each candidate gene and the <i>Bestkeeper</i> index is described by the Pearson correlation coefficient (<i>r</i>) and the probability value (<i>p</i>), based on all candidate genes (n = 9) or after exclusion of <i>GAPDH</i> and <i>B2M</i> (n = 7). Abbreviations: Ar. Mean: arithmetic mean; Geo. Mean: geometric mean; BK: <i>Bestkeeper</i> index.</p

Gene expression variability of 'universal reference genes' in human cells and diseases.

<p>Gene expression of previously reported universally stably expressed reference genes was monitored across 5372 human tissues samples representing 369 tissues and cell types or diseases clustered into 15 meta-groups (indicated on top of figure). Red and blue arrows indicate expression significantly above (Up) or below (Down) median expression within meta-group, respectively. p-values ranged from 10⁻² to less than 10⁻¹⁰. <b>Panels: A.</b> ARL8B; <b>B.</b> CTBP1; <b>C.</b> CUL1; <b>D.</b> DIMT1L; <b>E.</b> FBXW2; <b>F.</b> GPBP1; <b>G.</b> LUC7L2; <b>H.</b> OAZ1; <b>I.</b> PAPOLA; <b>J.</b> SPG21; <b>K.</b> TRIM27; <b>L.</b> UBQLN1; <b>M.</b> ZNF-207.</p