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

Nuclear receptor NR1I2, the pregnane X receptor (PXR; also known as the steroid and xenobiotic receptor, SXR), and the related NR1I3, the constitutive androstane receptor (CAR) are ligand-activated transcription factors often referred to as “xenobiotic sensors” (eg, Kretschmer and Baldwin, 2005; Timsit and Negishi, 2007; Willson and Kliewer, 2002). The aryl hydrocarbon receptor (Ahr) also is a ligand-dependent transcription factor that in vertebrates is activated by xenobiotics such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds (Fernandez-Salguero et al., 1996; Safe, 1990). Together, these receptors act to protect organisms from exogenous and endogenous toxic chemicals by regulating genes involved in xenobiotic metabolism and elimination, including cytochrome P450 (CYP) genes, various transferases, and ABC transporters (Kliewer et al., 2002; Kohle and Bock, 2009; Zhang et al., 2008). The extent to which these receptors are governing responses to different chemicals is not understood. Mammalian PXR ligands include a suite of environmental pollutants, drugs, and endogenous compounds such as bile salts and steroids (eg, Ekins et al., 2007; Kretschmer and Baldwin, 2005; Zhang et al., 2008). In cell-based assays, human PXR is activated by a large majority (234 of 320) of the EPA’s ToxCast_320 environmental substances (Martin et al., 2010). However, the ligand selectivity of PXR differs markedly among mammals. Thus, human PXR is strongly activated by rifampicin but not pregnenolone-16α-carbonitrile (PCN), whereas rodent PXR is strongly activated by PCN but not rifampicin (Jones et al., 2000; Moore et al., 2002). Similarly, clotrimazole (CLO) is an agonist for human but not mouse PXR (Moore et al., 2002). Such dramatic species differences in PXR activation have important implications for understanding effects of agonists in comparative physiology, and for the use of animal models to predict human risk. The CAR gene diverged from PXR during the course of vertebrate evolution; however, CAR was lost from or arose after divergence of the teleost fish line (Mathas et al., 2012). Thus, teleost fish such as zebrafish do not have CAR, magnifying the potential role of PXR in xenobiotic responses in these fish. In general, narrower selectivity has been observed for fish PXR (Ekins et al., 2008; Krasowski et al., 2011; Milnes et al., 2008; Moore et al., 2002) than for human PXR, and fish PXR appears to differ from the mammalian orthologs in endogenous ligand specificity (Ekins et al., 2008; Reschly et al., 2007). These observations are derived from cell-based reporter systems, but the ligand efficacy and transcriptional landscape of Pxr in vivo in zebrafish are unknown. Induction of rodent CYP2s and CYP3s has long been known to occur through the action of PXR and CAR (Waxman, 1999). Recently, we and others reported that chemicals identified as PXR agonists in cell-based in vitro studies were able to induce some CYP2 and CYP3A genes in fish (Bresolin et al., 2005; Kubota et al., 2013); however, the mechanism underlying chemical induction of fish CYP2 and CYP3 genes is not yet understood. Moreover, while some genes, eg, CYP3A65, are candidate targets of zebrafish Pxr, based on orthology to the PXR-regulated CYP3A genes in mammals, studies suggest that other transcription factors may be involved as well. Thus, zebrafish Ahr is responsible for dioxin and non-ortho-polychlorinated biphenyl induction of CYP1 family target genes (Handley-Goldstone et al., 2005; Jonsson et al., 2012; Kubota et al., 2011), but studies have shown that TCDD also can induce CYP3A65 in zebrafish (Chang et al., 2013; Tseng et al., 2005). Whether the Ahr is directly involved in regulating the expression of genes that also are regulated by Pxr in zebrafish is not known. Expanding our knowledge of the role of Pxr in regulating the expression of genes in vivo, and of the nature of cross-regulation of genes by Ahr and Pxr, is critical to establishing a mechanistic foundation for understanding and screening for chemical effects in this premiere toxicological model. We recently reported cloning, expression, and activation of full-length Pxr in zebrafish (Bainy et al., 2013). Here we address the role of zebrafish Pxr and Ahr2 in regulation of target CYP genes in vivo in developing zebrafish. Pxr involvement in response to agonists was established using morpholino antisense oligonucleotides (MO) to knock down translation of pxr, coupled with a search for putative Pxr response elements in proximal promoters of target genes. We also examined the role of Ahr2 in regulating the expression of the same genes examined for Pxr regulation, seeking cross-talk between the Pxr and Ahr in zebrafish.