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

Activation of CAR by an endogenous or foreign substance leads to translocation of the CAR-RXR heterodimer into the nucleus, and this dimer binds to DNA recognition elements in the regulatory region of CAR-responsive genes. CAR activation thus alters gene expression and upregulates xenobiotic-metabolizing enzymes such as CYP2B, CYP2C, CYP3A, sulfotransferases, UDP-glucuronyltransferases and glutathione transferases, as well as xenobiotic transporters such as Mrp2 and Mrp4 (Omiecinski et al., 2011a). In addition, CAR alters genes involved in lipid homeostasis, glucose utilization and energy metabolism. In rats and mice, the expression of additional genes involved in cell proliferation and apoptosis control are altered; Gadd45beta and Cdc20 are examples of genes that function in this way and are upregulated in mice within hours of treatment with a CAR activator (Peffer et al., 2018a;  Peffer et al., 2018b;  Tojima et al., 2012).

Activation of PXR (NR1I2), a related nuclear receptor to CAR (NR1I3), is a possible confounding factor that may be operative for certain substances. There is much cross-talk between CAR and PXR, and similar responsive genes, and a particular agent could produce a mixed set of gene expression response by activating both PXR and CAR (Tojima et al., 2012; Stanley et al., 2006).

A quantitative understanding supporting the linkage between CAR activation (the MIE) and measurable changes in the appropriate genes’ expression levels (KE1) is somewhat difficult to assess directly, because the methods to measure the actual event of CAR activation are largely confined to sub-molecular manipulations that are not conducive to in vivo dose-response assessment, or require in vitro techniques such as reporter assays, or nuclear translocation determinations (Chen et al., 2010;  Maglich et al., 2003;  Omiecinski et al., 2011b;  Stanley et al., 2006). Instead, assessments of the relationship between MIE and KE1 with CAR activators have been obtained via indirect demonstration that CAR activation has occurred, by examining  responses in both wild-type and CAR-null mice or rats, or by use of siRNA. As discussed above under "Empirical Evidence", data of this type for phenobarbital or TCPOBOP treatment in wild-type mice and CAR-null mice has demonstrated that changes in expression of CAR-responsive genes at a particular dose level in vivo requires the presence (and activation) of a functional CAR (Huang et al., 2005;  Peffer et al., 2007;  Tojima et al., 2012). Also, following metofluthrin treatment of rats, altered gene expression was observed (900 ppm and higher), and treatment of rat hepatocytes in vitro along with siRNA for CAR greatly attenuated the response of CAR-activated genes (Deguchi et al., 2009). Thus, there is a limited level of understanding of this quantitative relationship between MIE and KE1.

CAR receptors are present in the livers of virtually all mammalian species; however, there are important differences in protein sequence and thus ligand binding properties (Omiecinski et al., 2011b;  Reschly and Krasowski, 2006). In reporter assays for mouse, rat, dog and human CAR, clear qualitative as well as quantitative differences in the ability of suspected CAR activators to activate CAR from the different species were demonstrated (Omiecinski et al., 2011b). In terms of the specific KER of CAR activation directly leading to altered gene expression specific to CAR activation, in vitro hepatocyte experiments indicate that human hepatocytes have only partial overlap with mice and rats in terms of the genes that are affected. In particular, genes that are related to CYP induction (e.g. Cyp2b isoforms) show increases in expression across mouse, rat and human if the CAR molecule for that species is activated, but the pro-proliferative gene pathways  have been shown to be activated only in mice and rats (Elcombe et al., 2014;  Hasmall and Roberts, 1999;  Hirose et al., 2009;  Lake, 2009).  For example, the total number of altered genes in livers of chimeric mice that reflected human hepatocytes (293) compared to livers of similarly treated CD-1 mice (846) was much lower, and only 10 differentially expressed genes (primarily CYP genes) were common to both species’ liver samples following treatment with phenobarbital at dose levels of 1000 – 2500 ppm in the diet (Yamada et al., 2014).