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. 2017 Apr;58(4):576-585.
doi: 10.1111/epi.13703. Epub 2017 Feb 15.

Overexpression of pregnane X and glucocorticoid receptors and the regulation of cytochrome P450 in human epileptic brain endothelial cells

Chaitali Ghosh  1   2 , Mohammed Hossain  1 , Jesal Solanki  1 , Imad M Najm  3 , Nicola Marchi  4 , Damir Janigro  5   6
Affiliations

Overexpression of pregnane X and glucocorticoid receptors and the regulation of cytochrome P450 in human epileptic brain endothelial cells

Chaitali Ghosh et al. Epilepsia. 2017 Apr.

Abstract

Objective: Recent evidence suggests a metabolic contribution of cytochrome P450 enzymes (CYPs) to the drug-resistant phenotype in human epilepsy. However, the upstream molecular regulators of CYP in the epileptic brain remain understudied. We therefore investigated the expression and function of pregnane xenobiotic (PXR) and glucocorticoid (GR) nuclear receptors in endothelial cells established from post-epilepsy surgery brain samples.

Methods: PXR/GR localization was evaluated by immunohistochemistry in specimens from subjects who underwent temporal lobe resections to relieve drug-resistant seizures. We used primary cultures of endothelial cells obtained from epileptic brain tissues (EPI-ECs; n = 8), commercially available human brain microvascular endothelial cells (HBMECs; n = 8), and human hepatocytes (n = 3). PXR/GR messenger RNA (mRNA) levels in brain ECs was initially determined by complementary DNA (cDNA) microarrays. The expression of PXR/GR proteins was quantified by Western blot. PXR and GR silencing was performed in EPI-ECs (n = 4), and the impact on downstream CYP expression was determined.

Results: PXR/GR expression was detected by immunofluorescence in ECs and neurons in the human temporal lobe samples analyzed. Elevated mRNA and protein levels of PXR and GR were found in EPI-ECs versus control HBMECs. Hepatocytes, used as a positive control, displayed the highest levels of PXR/GR expression. We confirmed expression of PXR/GR in cytoplasmic-nuclear subcellular fractions, with a significant increase of PXR/GR in EPI-ECs versus controls. CYP3A4, CYP2C9, and CYP2E1 were overexpressed in EPI-ECs versus control, whereas CYP2D6 and CYP2C19 were downregulated or absent in EPI-ECs. GR silencing in EPI-ECs led to decreased CYP3A4, CYP2C9, and PXR expression. PXR silencing in EPI-ECs resulted in the specific downregulation of CYP3A4 expression.

Significance: Our results indicate increased PXR and GR in primary ECs derived from human epileptic brains. PXR or GR may be responsible for a local drug brain metabolism sustained by abnormal CYP regulation.

Keywords: Drug resistance; Epilepsy; Neurovascular unit; Nuclear Receptors.

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Figures

Figure 1
Figure 1
Expression of PXR, GR, and CYP3A4 in human epileptic brains. (A–C1) NRs were found at the blood–brain barrier interface and in neurons in epileptic brains (n = 5). Magnification of gliotic regions (**) and GFAP+ staining on a resected human epileptic brain showed increased expression of NRs (PXRs, GRs), and CYP3A4. This staining pattern was less evident in nongliotic regions (*). Note: The indicators for neurons are depicted as arrowheads and those for capillaries as yellow arrows. Co‐localization was evaluated with neuronal markers (NeuN, A,B) and glial markers (GFAP, A1–B1 and C–C1). (A–A2) PXR expression was observed at the vascular interface (A–A1, yellow arrows) and in neurons (A and A2). (B–B2) A similar pattern was found for GR expression across the epileptic brain slices. (A2,B2) Co‐localization of GR or PXR staining with CYP3A4 was found in regions of reactive gliosis (**). (C,C1) Representative images of brain samples from patients with temporal lobe epilepsy show GR staining in regions of reactive gliosis (**).
Figure 2
Figure 2
PXR and GR expression patterns in human EPI‐ECs. (A) The mRNA levels of NRs (human retinoid x receptor, RXR; aryl hydrocarbon receptors, AHRs; glucocorticoid receptors, GRs); and orphan receptors [e.g., pregnane X receptors, PXRs]) in EPI‐ECs were significantly increased (*p < 0.05, n = 4) compared to controls (HBMECs). (B–B2) PXR and GR protein expression evaluated by Western blot showed variability among the samples analyzed; increased levels of PXRs (*p < 0.05) and GR (*p < 0.05) were found in EPI‐ECs compared with HBMECs (B1–B2) (n = 8). Representative Western blots are shown in B for PXR/GR (n = 4/each, HBMEC or EPI‐ECs). The hepatocytes (HEPATO) consistently showed an increased level (**p < 0.01, n = 3) of PXRs and GRs as compared to EPI‐ECs. Glyceraldehyde 3‐phosphate dehydrogenase and β‐actin were used as loading controls for mRNA and protein, respectively. Results are expressed as mean ± standard error of the mean (SEM) (analysis of variance, ANOVA). (C–C2) Western blot results increased levels of PXR and GR in the cytoplasmic and nuclear (*p < 0.05, n = 4) EPI‐ECs fractions. β‐Actin was used as a loading control for cytoplasmic fractions; proliferating cell nuclear antigen (PCNA) was used for nuclear fractions. Results are expressed as mean ± SEM (ANOVA).
Figure 3
Figure 3
Overexpression of specific CYPs and correlation with PXR and GR protein levels in EPI‐ECs. (A) Western blot showing elevated expression of CYP3A4, CYP2C9, and CYP2E1 in EPI‐ECs (1 to 4), compared to HBMECs (1 to 4). In contrast, CYP2D6 and CYP2C19 were down‐regulated or absent. Quantitative analysis of protein expression and correlation of PXR/GR with CYP levels showed that CYP3A4, CYP2E1, and CYP2C9 levels increased significantly in accordance with increases in PXR (B) and GR (D) expression in EPI‐ECs. CYP2C19 and CYP2D6 showed decreased levels in correlation with decreased levels of PXR (C) or GR (E) expression in EPI‐ECs compared with HBMECs (n = 4/each for HBMECs or EPI‐ECs, in triplicate). Results are expressed as mean ± SEM (ANOVA) and were normalized with β‐actin as loading control.
Figure 4
Figure 4
GR in human EPI‐ECs modulates expression of CYP3A4, CYP2C9, and PXR. (A) GR silencing of genes in EPI‐ECs was evaluated by Western blot, also showing a corresponding decrease in CYP3A4 and CYP2C9 expression. CYP2E1 expression did not change. GR silencing in EPI‐ECs also showed a corresponding decrease in PXR expression. (B) CYP3A4, CYP2E1, and CYP2C9 levels in GR‐silenced (siRNA) EPI‐ECs are compared to nontransfected EPI‐ECs (non‐siRNA), in which GR silencing influenced CYP3A4 and CYP2C9 expression. (C) The quantitative plots of the CYP3A4, CYP2E1, and CYP2C9 levels in GR siRNA and non‐siRNA EPI‐ECs showed that the levels of CYP3A4‐GR and CYP2C9‐GR are correlated, and siRNA GR/CYPs in EPI‐ECs levels are comparable to those of control brain endothelial cells (HBMECs). Such correlation was not applicable for CYP2E1‐GR (n = 4 for each EPI‐EC siRNA or non‐siRNA or HBMECs, in triplicate). β‐Actin was used as a loading control and normalization. Results are expressed as mean ± SEM (ANOVA).
Figure 5
Figure 5
Differential involvement of PXR and CYPs in EPI‐ECs. (A) Western blot showing PXR silencing and resulting CYP3A4, CYP2C9, CYP2E1, and GR protein expression in EPI‐ECs. The PXR siRNA (denoted by EPI‐ECs 1 to 4) and non‐siRNA counterparts in EPI‐ECs were compared individually. PXR silencing showed negligible alternation in CYP2C9 and GR expression. The dotted line shows blots that were performed separately. (B) CYP3A4, CYP2E1, and CYP2C9 levels in PXR silenced (siRNA) EPI‐ECs and nontransfected EPI‐ECs (non‐siRNA EPI‐ECs) were quantified. (C–C2) CYP3A4, CYP2E1, and CYP2C9 levels corresponding to PXR silencing (or not) in EPI‐ECs and compared to control ECs (n = 4/EPI‐EC siRNA or non‐siRNA or HBMECs). β‐Actin was used as a loading control and normalization. Results are expressed as mean ± SEM (ANOVA).
Figure 6
Figure 6
Schematic representation of potential PXR‐GR and CYP regulation in human EPI‐ECs. (A) Pathophysiologic factors, drugs, and disease etiology may affect the basal expression levels of PXR and GR in EPI‐ECs. These, in turn, could regulate specific CYPs levels (CYP3A4, CYP2E1, and CYP2C9), possibly partaking to drug resistance in epilepsy. (B) The PXR/GR expression pattern and CYP regulation in EPI‐ECs suggests that GRs could possibly be a master controller of PXRs (Fig. 4 and as described earlier in hepatocytes5). GRs directly regulate CYP3A4, CYP2C9, and PXR expression in EPI‐ECs.
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