Abstract
The pregnane X receptor (PXR) is known as the xenosensing receptor responsible for coordinated regulation of metabolic genes in response to diverse xenobiotic challenges. In particular, the ability of the PXR to regulate CYP3A4, the enzyme capable of metabolizing more than 60% of all pharmaceuticals, defines its metabolic importance. Currently the list of PXR ligands and target genes is extensive, yet investigations into the regulation and expression of PXRs are few. After an initial review of available sequence data, we discovered discrepancies in the 5′ untranslated region (UTR) and transcriptional start site (TSS) characterizations of the human PXR gene and subsequently endeavored to define TSSs and proximal promoters for isoforms PXR 1 and PXR 2. Reverse transcriptase-polymerase chain reaction and primer extension experiments performed on RNA from human liver identified two TSSs for each receptor isoform. These results extended the 5′UTR sequence of each isoform and defined new proximal promoters for both. Candidate response elements for liver-enriched transcription factors and other receptors were found in both proximal promoters. Quantitative PCR from human liver illustrated a highly variable expression profile for total PXRs; yet PXR 2 expression represented a consistent 2 to 5% of total PXR expression, despite the observed variability. Transfection experiments demonstrated that PXR 1 and PXR 2 had comparable abilities to transcriptionally activate the CYP3A4 promoter. Collectively, comparable function, consistent expression, and independent regulation suggest that PXR 2 is capable of contributing to the cumulative function of PXRs and should be included in the larger investigations of PXR expression and regulation.
Footnotes
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The data presented here form part of the dissertation of Leslie M. Tompkins that was submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.018317.
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ABBREVIATIONS: PXR, pregnane X receptor; P450, cytochrome P450; GR, glucocorticoid receptor; PPAR, peroxisome proliferator activated receptor; TSS, transcription start site; RT, reverse transcription; PCR, polymerase chain reaction; kb, kilobase(s); nt, nucleotide(s); RU486, 17β-hydroxy-11β-[4-dimethylamino phenyl]-17α-[1-propynyl]estra-4,9-dien-3-one; PBDE-71, polybrominated diphenyl ether-71; ANOVA, analysis of variance; NCBI, National Center for Biotechnology Information; bp, base pair(s); PR, progesterone receptor; 5′UTR, 5′ untranslated region.
- Received August 15, 2007.
- Accepted February 12, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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