Upstream eventActivation, Constitutive androstane receptor
Altered gene expression specific to CAR activation, Hepatocytes
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding|
|Constitutive androstane receptor activation leading to hepatocellular adenomas and carcinomas in the mouse and the rat||adjacent||High||Moderate|
Life Stage Applicability
Key Event Relationship Description
The constitutive androstane receptor (CAR; NR1I3) is a nuclear receptor that is expressed primarily in the liver, which can be activated by xenobiotics or by certain endogenous cellular metabolites. CAR normally is tethered in the cytoplasm of a hepatocyte via a set of specific proteins including heat shock protein 90 (HSP90) and other chaperones. Chemical ligands bind to the ligand binding site of CAR, and a conformational change frees CAR from the tethering proteins and facilitates its transport into the nucleus. In addition, indirect CAR activators (e.g. phenobarbital) can bind to the EGF receptor to initiate a series of steps that eventually dephosphorylate a critical Threonine-38 residue in CAR, allowing it to migrate into the nucleus. Inside the nucleus, CAR dimerizes with RXR and this CAR-RXR complex binds to specific response elements on the DNA to activate transcription of specific CAR-responsive genes. CAR is unique among nuclear receptors, in that it is constitutively active when in the nucleus, i.e. it will spontaneously dimerize with RXR and alter gene expression, even without an activator bound to its ligand binding domain. When activated and translocated to the nucleus, CAR alters the transcription of multiple genes, which can be characterized as falling into three general areas of biological function: 1) Phase I and II metabolizing enzymes plus transporters; 2) decreases in lipogenesis and gluconeogenesis enzymes; and 3) species-specific alteration of cell proliferation and apoptosis signals (Mutoh et al., 2013; Omiecinski et al., 2011; Elcombe et al., 2014).
In terms of this AOP, CAR activation in rat or mouse hepatocytes directly leads to altered expression of genes that produce an intracellular signal for increased cell proliferation.
Evidence Supporting this KER
Activation of the CAR receptor has been demonstrated to produce increases in CAR-responsive genes such as Cyp2b10 (mice), Cyp2b1/2 (rats), Gadd45b and Cdc20 and alterations in a series of genes that give an overall pathway change associated with increased progression through the cell cycle (Deguchi et al., 2009; Geter et al., 2014; Ross et al., 2010; Tojima et al., 2012). 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.
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).
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 (NextBio.com) or via examination IPA software 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 (-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).
Uncertainties and Inconsistencies
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 (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.
Quantitative Understanding of the Linkage
The quantitative understanding supporting the linkage between CAR activation and measurable changes in the appropriate genes’ expression levels has been established in a limited number of studies with ample dose-response data in mice (Geter et al., 2014) and in rats (Deguchi et al., 2009). In other examples (e.g. TCPOBOP in mice), the key event of altered gene expression characteristic of CAR activation (including Cyp2b10 and pro-proliferative / anti-apoptotic signaling genes) was measured at a known tumorigenic dose, but a full range of dose levels and responses was not available (Huang et al., 2005; Tojima et al., 2012; Yamamoto et al., 2004). Thus, there is a Moderate level of understanding of this quantitative relationship.
Known modulating factors
Known Feedforward/Feedback loops influencing this KER
Domain of Applicability
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. 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., 2011). In terms of the specific KER of CAR activation directly leading to altered gene expression specific to CAR activation, in vitro hepatocyte experiments indicates that human hepatocytes (and those of other species such as hamster) 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 (Cyp2b isoforms) show increases in expression across all species if the CAR molecule for that species is activated, but the pro-proliferative gene pathways appear to be only differentially expressed in mice and rats (Elcombe et al., 2014; Hasmall and Roberts, 1999; Hirose et al., 2009; Lake, 2009).
[see reference list at end of this AOP; it includes all cited references]