PXR IN BRAIN: NOVEL METHODS TO CHARACTERIZE ITS BIOLOGY

Abstract

Pregnane X receptor (PXR, NR1I2) belongs to the nuclear receptor superfamily of ligand-activated transcription factors and was identified in 1998. PXR is highly expressed in the liver and intestine, and is involved in regulating the expression of genes that encode important drug metabolizing enzymes as well as several key drug transporter proteins. Previous studies have found that ligand-mediated activation of PXR can increase the expression of multidrug resistance protein 1 (Mdr1) in the endothelial cells in the intestine in mice. However, there is no correlation between PXR activation and Mdr1 gene expression levels in liver hepatocytes in these animals. Thus, PXR biology exhibits a curious phenomenon in that there appears to be a tissue-specific role of this nuclear receptor superfamily member. Currently, the molecular mechanism(s) underlying this phenomenon are not known, but we hypothesize here that tissue-specific PXR-binding and co-regulatory/accessory proteins likely play a key role in governing this phenomenon. While liver and intestine express high levels of PXR, other tissues have been found to express lower but significant levels of this nuclear receptor protein including kidney, ovary, stomach, and brain. Several lines of evidence support the notion of a key role for PXR in regulating brain function. First, the expression of PXR was identified in 2004 in brain capillary endothelial cells. Lower levels of PXR were also detected in several brain regions of different species including rat, rabbit, pig, human, and mouse. Several key endogenous neurosteroidal compounds, including allopregnanolone, have been demonstrated to serve as PXR-ligands to dramatically increase the trans-activation capacity of this nuclear receptor family member. Finally, the proestrous rats infused with PXR anti-sense oligonucleotides to the ventral tegmental area significantly decreased levels of allopregnanolone, further suggesting an interface between PXR and allopregnanolone metabolism. Therefore, the purpose of the research described in this thesis is (1) to develop a method for the identification of tissue-specific PXR-binding proteins, and (2) to characterize the potential effects of PXR deletion and PXR activation on the expression of the genes that encode the rate limiting enzymes in the production of allopregnanolone in mice. In the first study, I developed a novel protocol using adenovirus-mediated methods coupled with primary cultures of rat hepatocytes and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify hepatocyte-specific PXR-binding proteins. In the second study, I tested the hypothesis that PXR can regulate 5α-reductase type 1 (Srd5α1) and type 2 (Srd5α2) gene expression levels in mouse brain. My results reveal that deletion of PXR in mice alters basal expression levels of Srd5α1 and Srd5α2 in a tissue-specific manner. Additionally, Pregnenolone 16α-carbonitrile (PCN) decreased Srd5α1 and Srd5α2 gene expression in the liver and several brain regions in both wild type and PXR knockout mice, suggesting that PCN-mediated decrease of Srd5α1 and Srd5α2 gene expression is in a PXR-independent manner. Taken together, the data presented in this thesis shed new light upon the role of PXR in regulating the expression of key target genes in the brain. In particular, the data suggest that while PXR plays a role in neurosteroid metabolism, there is also a key PXR-independent role in neurosteroid metabolism in several brain tissue types following exposure to steroidal compounds such as PCN. Finally, the biochemical procedures developed and validated in this thesis should be useful in identifying novel PXR-binding proteins from primary cultures of neuronal cells, as well as other cell types amenable to primary culture methods.