Activation, Constitutive androstane receptor leads to Altered expression of hepatic CAR-dependent genes

It is highly plausible that activation of CAR would produce changes in expression of specific genes, since transcription of genes specifically associated with CAR response elements is how this nuclear receptor achieves its biological effects (Tien and Negishi, 2006). In mice, rat and human hepatocytes, CAR activation produces changes in genes related to CYP2B and CYP3A enzymes, certain sulfotransferase and glucuronyltransferase enzymes, hepatic transport proteins and genes related to lipid and glucose metabolism (Maglich et al., 2003;  Omiecinski et al., 2011b;  Omiecinski et al., 2011a;  Tien and Negishi, 2006). In mice and rats, CAR activation has been shown to also achieve alterations in genes that give an overall pathway change associated with increased progression through the cell cycle such as Gadd45b and Cdc20 (Deguchi et al., 2009;  Geter et al., 2014;  Ross et al., 2010;  Tojima et al., 2012).

Strong support for this linkage comes from in vivo and in vitro studies with model CAR activators, and via the absence of the same effects including specific gene expression changes in CAR knockout mice lacking the CAR receptor. In a study with TCPOBOP (a direct activator of mouse CAR), these CAR-specific changes included Gadd45b (↑ 14-fold), plus Cdc20 (↑ 37-fold) and additional cytokines, and these genes were unaffected in CAR knockout mice (Tojima et al., 2012). Gene pathways (by Ingenuity Pathway Analysis, IPA) that were altered by phenobarbital in a dose-responsive manner in CD-1 mice included “Cell cycle of chromosomal replication” and “Cell cycle: G2/M DNA damage checkpoint regulation”; these were only significantly altered above a dose level of 15 mg/kg/day (Geter et al., 2014). In addition, CAR knockout mice showed absent or greatly diminished induction of mRNA (compared to wild-type mice) for Cyp2b10 after treatment with TCPOBOP or phenobarbital (Ross et al., 2010;  Tojima et al., 2012).

With humans, a selective activator of human CAR  (CITCO;  6-(4-Chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime) was discovered that has shown little or no ability to activate the corresponding CAR of mice and rats in CAR3 reporter assays, whereas it produced a strong positive response in reporter assays for human CAR (Maglich et al., 2003;  Omiecinski et al., 2011b). Upon treatment of human hepatocytes with CITCO or a model PXR activator (rifampicin), CITCO produced a change in expression of a suite of eight marker changes that was distinct from that of the PXR activator. The degree of fold-changes varied greatly across three different human donors, but consistent CAR-related changes were seen in CYP2B6, CYP3A4, CYP2A6, ALAS, ALDH1A4, and GSTA2, with induction in 1 or 2 of the donors for the genes MDR1 and SULT1A1 (Maglich et al., 2003).

Oshida et al. (2015a) have published a “CAR signature” that is based upon changes in 83 genes consistently expressed in mice with three different CAR activators (phenobarbital, TCPOBOP and CITCO). Comparison to this CAR biomarker signature for new molecules is possible via a running Fisher’s p-value in Correlation Engine (Illumina) or via examination IPA software (Ingenuity Pathway Analysis) of the 10 most altered pathways. An approach like this is useful to establish a CAR gene expression profile has occurred, since it relies on an overall pathway rather than a small number of selected genes. For example, the triazole fungicide cyproconazole gave a highly significant response compared to the CAR signature (-log [p-values] of 8.0, 12.4 and 19.0 for 100, 200 and 400 ppm doses), and the gene signature did not match similar published biomarker signatures for AhR or PPARα (Oshida et al., 2015a;  Oshida et al., 2015b;  Oshida et al., 2015c;  Peffer et al., 2018b).

In rats, metofluthrin has been shown to be a strong CAR activator in the liver. The key event of increased Cyp2b1/2 and/or Cyp3a1 mRNA expression levels was demonstrated at dose levels of 900 ppm and above, and they were unaffected at the NOAEL of 200 ppm in the diet. In isolated rat hepatocyte cultures treated with 50 µm metofluthrin, co-administration of siRNA for CAR (a gene silencing technique) caused a knockdown of Car mRNA expression and resulting suppression of the response to metofluthrin in terms of Cyp2b1 mRNA and Car mRNA levels (Deguchi et al., 2009), thus providing evidence for the MIE (CAR activation) and the downstream key event (altered gene expression secondary to CAR activation).

In other mammalian species such as dogs, hamsters, and non-human primates, the presence of a functional CAR receptor with a known species-specific amino acid sequence has been demonstrated (Omiecinski et al., 2011b;  Reschly and Krasowski, 2006), but very little experimentation with the gene expression effects of CAR activators in these species has been described thus far in the literature.

In general, CAR activators show very consistent, large fold-increases for the characteristic expression of Cyp2b isoforms across in vivo studies in multiple species and with many different molecules. While certain genes related to a pro-proliferative effect appear to be CAR-mediated and reproducibly impacted in multiple studies (Currie et al., 2014;  Deguchi et al., 2009;  Geter et al., 2014;  Peffer et al., 2007;  Tojima et al., 2012), there are examples where changes in a specific gene was not observed. For example, Ross et al. (2010) tested 80 mg/kg/day (ip dosing) phenobarbital for 4 days in WT C57BL/6J mice, and they observed a 15.8-fold increase in Cdc20, but did not see an increased expression of Gadd45b. Mapping of a specific genes’ changes following activation of CAR by a particular CAR activator may be affected by the species, strain, dose level and time point examined, as well as the other non-CAR effects of that molecule. Examining for a significant pathway change is likely to be a more reliable measure of this Key Event Relationship (Oshida et al., 2015a), but this is also somewhat dependent on the experimental design, the species and duration of treatment, and the pathway analysis tools.

Because the extent or degree of activation of CAR is not readily measurable with in vivo studies, the nature of the relationship between CAR activation and altered mRNA expression (e.g. linear, exponential, other) cannot be stated. In addition, the direction of change in certain CAR-responsive genes is variable, in that CAR activation causes an increase in expression of some genes and a decrease in expression of other genes. For example, in mice treated with TCPOBOP by ip injection, Tojima et al. (2012) showed large increases in expression of Cyp2b10 (151x), Cdc20 (37x) and Gadd45b (14x), plus decreases in expression of lipid-related genes Acsl5 (0.5x), Slc21a1 (0.2x) and Hmgcs2 (0.3x). The different fold-change values for specific genes also indicates that the magnitude of the gene expression differences can be very dependent on the properties of the individual gene, and whether it is normally active or quiescent prior to treatment with a CAR activator.

The onset of CAR activation (upstream KE) and the time scale of measurable differences in gene expression (downstream KE) are influenced by variables such as pharmacokinetics and route of administration of an administered stressor. However, it is established that exposure of the livers of mice to a CAR activator (TCPOBOP) by intraperitoneal injection, thus bypassing the intestinal transit and absorption processes, was able to achieve large changes in gene expression in the liver after 12 hours (Tojima et al., 2012). The cascade of normal biological events following CAR activation by a ligand (i.e. binding, nuclear translocation, altered gene expression, and post-transcriptional modifications of mRNA) likely takes several hours to be completed, after which a measurable difference vs. control in mRNA levels is detectable by RT-PCR or microarrays. After the initial perturbation by a stressor of CAR, the time course of changes in gene expression is indefinite. That is, some gene expression changes can continue to be observed for as long as the CAR activator is present. For example, in mice treated with phenobarbital for up to 32 weeks following initiation with a single dose of diethylnitrosamine, differences in gene expression of CAR-responsive genes such as Gadd45b and Cdc2 continued to be observed throughout the entire treatment period in both non-neoplastic liver tissue and in liver tumors of treated vs. untreated animals (Phillips et al., 2009a).

In terms of this specific KER1 of activation of CAR (MIE) leading directly to altered expression of CAR-responsive genes (KE1), there are no known feedback loops that would affect this overall process.  As an example of this lack of feedback alteration, when mice were treated long-term with phenobarbital, they continued to show significant changes in expression of CAR-responsive genes up through 32 weeks of treatment (Phillips et al., 2009).