Role of Pregnane X Receptor and Aryl Hydrocarbon Receptor in Transcriptional Regulation of pxr, CYP2, and CYP3 Genes in Developing Zebrafish

Abstract

Ligand-activated receptors regulate numerous genes, and mediate effects of a broad set of endogenous and exogenous chemicals in vertebrates. Understanding the roles of these transcription factors in zebrafish (Danio rerio) is important to the use of this non-mammalian model in toxicological, pharmacological, and carcinogenesis research. Response to a potential agonist for the pregnane X receptor (Pxr) [pregnenolone (PN)] was examined in developing zebrafish, to assess involvement of Pxr in regulation of selected genes, including genes in cytochrome P450 subfamilies CYP2 and CYP3. We also examined interaction of Pxr and the aryl hydrocarbon receptor (Ahr) signaling pathways. Pregnenolone caused a dose-dependent increase in mRNA levels of pxr, ahr2, CYP1A, CYP2AA1, CYP2AA12, CYP3A65, and CYP3C1, most of which peaked at 3 µM PN. The well-known Ahr agonist 3,3',4,4',5-pentachlorobiphenyl (PCB126) also upregulated expression of pxr, ahr2, CYP1A, CYP2AA12, CYP3A65, and CYP3C1 in a dose-dependent manner. Inhibition of pxr translation by morpholino antisense oligonucleotides (MO) suppressed PN-induced expression of pxr, ahr2, CYP3A65, and CYP3C1 genes. Levels of CYP2AA1 and CYP2AA12 mRNA were increased in the control-MO group exposed to PN; this was prevented by knocking down Pxr. Similarly, Ahr2-MO treatment blocked PCB126-induced mRNA expression of pxr, CYP1A, CYP2AA12, CYP3A65, and CYP3C1. The present study shows self-regulation of pxr by PN in developing zebrafish. Selected zebrafish CYP1, CYP2 (including several CYP2AAs) and CYP3 genes appear to be under the regulation of both Pxr and Ahr2.

Integrated in silico and in vivo approaches to investigate effects of BDE‐99 mediated by the nuclear receptors on developing zebrafish

“…Cross-talk between PXR and AhR signaling pathways was recently demonstrated in zebrafish (Kubota et al 2015), and was also suggested in mammals (Maglich et al 2002). This cross-talk in zebrafish was considered reciprocal rather than asymmetric, because ahr2 activation caused up-regulation of pxr, cytochrome P450 proteins 2 (cyp2), and cytochrome P450 proteins 3 (cyp3) genes, and PXR activation upregulated ahr2 and cyp1a1.…”

Regulation of pregnane-X-receptor, CYP3A and P-glycoprotein genes in the PCB-resistant killifish (Fundulus heteroclitus) population from New Bedford Harbor

“…In human hepatocytes, activation of PXR leads to induction of AhR-CYP1A1/CYP1A2 signaling (Maglich et al, 2002). Cross-talk between AhR and PXR was recently described in zebrafish (Kubota et al, 2014). The NBH killifish have an AhR-CYP1A signaling with reduced sensitivity to PCB exposure, but these fish responded to PCB treatment by induction of PXR, CYP3A and Pgp mRNA levels in liver.…”

CRISPR-Cas9 mutated pregnane x receptor (pxr) retains pregnenolone-induced expression of cytochrome p450 family 3, subfamily A, polypeptide 65 (cyp3a65) in zebrafish (Danio rerio) larvae

“…We predicted that mutations would result in premature translational termination, eliminating function. In physiological terms we expected the mutations to recapitulate the loss-of-function outcomes observed from morpholino knockdown studies in zebrafish (Kubota et al, 2015) and genetic knockout studies in rodents (Xie et al, 2000), essentially abrogating canonical ligand-activated induction of target genes, such as PXR and CYP3A. Based on current zebrafish pxr gene models, four full-length transcripts have been identified containing identical exons 2 -8 with variability detected in exons 1, 9 and 10.…”

“…Notably, even among mammals PXR exhibits differences in ligandmediated activation, for example, the human PXR agonist rifampicin does not activate rat PXR, while pregnane-16α-carbonitrile (PCN), a known rat PXR agonist, fails to activate human PXR (Igarashi et al, 2012;Tirona et al, 2004;Xie et al, 2000). Notably, however, the steroid pregnenolone (PN) is a broadly conserved ligand across vertebrates, including zebrafish Kubota et al, 2015).…”