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

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.

β-glucuronidase mRNA levels are correlated with gait and working memory in premutation females: understanding the role of FMR1 premutation alleles

“…Moreover, GUS /2IC was significantly negatively correlated with FMR1 / GUS which again suggests that using GUS as a sole internal control gene for the quantification of FMR1 mRNA introduces variability into the data that is not consistent with changes in FMR1 mRNA levels. As recently highlighted, this is not a problem unique to the FXSD field, and there is growing recognition that other factors, such as inflammation, can significantly influence expression of internal control genes across various tissues, cell types and clinical manifestations 34 . Indeed, the present study identified that working memory performance in PM females was significantly correlated with both GUS mRNA levels and the ratio between the mRNA levels of EIF4A2 and SDHA used to normalize GUS , indicating that GUS is not likely to be the only gene dysregulated in PM females with working memory deficits.…”

Markers and Biomarkers of Endothelium: When Something Is Rotten in the State

“…RLIP76/RALBP1 RLIP76 (Ral-interacting protein of 76 kDa), also known as RalBP1 (Ral-binding protein 1), is an ATP-dependent transporter of electrophile-glutathione conjugates [56].…”

“…Inflammation modulates gene expression through the activation of NF-κB and other transcription factors. Mediators of inflammation can influence BBB permeability through RLIP76 (also known as RALBP1), an ATPdependent transporter of electrophile-glutathione conjugates [56]. The adherens junction is a principal component of intercellular adhesion and is comprised of transmembrane cadherins forming homotypic interactions between adjacent cells and cytoplasmic catenins linking the cadherins to the cytoskeleton.…”

Pathogenic Triad in Bacterial Meningitis: Pathogen Invasion, NF-κB Activation, and Leukocyte Transmigration that Occur at the Blood-Brain Barrier

“…It is also a transcriptional activator of many genes involved in the pathogenic triad of BM (e.g., IL–1β, TNF–α, IL-6, IL-8). During the past several decades, evidence has accumulated that deregulated activity of this pathway has been linked to the progression of a number of human diseases, including cancer, BM, rheumatoid arthritis, streptococcal cell wall-induced arthritis, experimental colitis, septic shock, Alzheimer's disease, multiple sclerosis, traumatic CNS injury, and cerebral ischemia (Koedel et al, 2000 ; de Souza et al, 2011 ; Anthony Jalin et al, 2015 ; Bennani-Baiti et al, 2015 ; Yu et al, 2015 ). Therefore, the NF-kB signaling pathway should be a good drug target that can hasten development of novel therapeutic agents.…”