Pregnane-X receptor (PXR; encoded by mammalian NR1I2 gene) is a nuclear receptor (NR) that regulates transcriptional responses to certain drugs and environmental toxicants — in part by inducing transcription of one or more cytochrome P450 3A enzymes that are found in most vertebrate species. PXR is activated by a wide range of ligands that differ across species, making functional studies on its role in the animal’s defense system problematic — when approached in a species-specific manner. Mammalian CYP3A-knockout studies have shown a requirement for PXR in the ligand-dependent activation of CYP3A expression (or reporter-gene activity). Morpholino knockdown (the structure of a morpholino oligomeric molecule has DNA bases attached to a backbone of methylenemorpholine rings, linked through phosphorodiamidate groups; sequences are designed to modify, or suppress, mRNA expression of a specific gene) of Pxr expression in zebrafish has indicated a requirement similar to that in mammals.
Pregnenolone, which binds to and activates PXR, is a naturally-occurring steroid hormone in humans that participates in production of other hormones — such as progesterone and estrogen. Some patients are prescribed pregnenolone to “improve their memory.” Side effects include drowsiness; thus, some patients take this hormone as an anti-anxiety drug. In other patients, however, pregnenolone increases anxiety. Pregnenolone is also an environmental contaminant, detected in most bodies of water in the U.S. because — as with virtually all drugs — people tend to flush unneeded drugs down the toilet. ☹
Authors [see attached article] describe herein generation of two zebrafish lines — each carrying a heritable deletion in the Pxr coding region. Both lines were predicted to result in the loss of a functional gene product. The zebrafish Pxr genomic gene spans ~65 kilobases (65,000 base-pairs [bp]) on chromosome 9, and “contains 9 or 10 exons” with the coding sequence (i.e. segment of DNA, transcribed into the mRNA, that is translated into the functional protein) located in the first nine exons.
One zebrafish line carried a 108-bp deletion in exon 2 (which includes sequence for the DNA-binding domain [DBD] and results in a frameshift defect) and, subsequently, an early termination codon (that can be detected in the expressed transcript). The second zebrafish line contained a 236-bp deletion, including a 37-bp deletion in exon 7, plus total removal of intron 7 and exon 8; the expressed transcript — from the “exon 7-8 mutant” allele — revealed direct splicing of exon 6 to exon 9. Larvae of these zebrafish lines were exposed to the established PXR ligand, pregnenolone, and authors looked for induction of Cyp3a65 mRNA expression.
Unexpectedly [see attached article], authors determined that pregnenolone-treated zebrafish larvae — homozygous (i.e. both gene copies are the same) for either of the Pxr mutant alleles — retained their ability to induce Cyp3a65 mRNA expression. In other words, zebrafish carrying Pxr mutant alleles, having deletions in either the DNA-binding domain or the ligand-binding domains, did NOT result in the (expected) loss-of-function (LoF) phenotype. This finding suggests that a compensatory mechanism might be responsible for Cyp3a65 induction. A second possibility is that PXR is not required for activating selected genes such as Cyp3a65 induction, A third (remote) possibility is that these truncated transcripts of the mutant Pxr lines are somehow still sufficient for other downstream transcriptional effects of PXR. Authors state that “this study highlights the potential for compensatory mechanisms — in the intact animal — to ‘avoid deleterious effects’ caused by serious gene mutations.” 😊
DwN
Toxicol Sci Mar 2020; 174: 51–62