Characterization of Constitutive Androstane Receptor (CAR) Activation

As the closest relative of the aforementioned PXR, the constitutive androstane/activator receptor (CAR, NR1I3) also governs the transcription of numerous hepatic drug-metabolizing enzymes and transporters in response to various xenobiotic exposures. Unlike most prototypical nuclear receptors, however, CAR can be activated via both direct ligand-binding and ligand-independent indirect mechanisms. Moreover, whereas CAR predominantly resides in the cytoplasm of primary hepatocytes in the absence of chemical stimulation, it simultaneously localizes in the nucleus of nearly all immortalized cell lines and is constitutively activated without chemical activation, making in vitro identification of CAR activators extremely challenging. In this chapter, we provide detailed step-by-step instructions for the application of two recently developed in vitro human (h) CAR activation assays: the hCAR1 + A-based luciferase assay in HepG2 cells and the adenovirus-based hCAR translocation assay in human primary hepatocytes. In combination, these assays are efficient in the identification of both direct and indirect activators of hCAR in vitro .