High-Content Analysis of Constitutive Androstane Receptor Nuclear Translocation.

Abstract

The constitutive androstane receptor (CAR; NR1I3) is a xenobiotic receptor that upregulates metabolism and detoxification mechanisms in the liver in response to chemical stimulation. Drug-induced activation of CAR may result in clinically significant drug-drug interactions and lead to complicated therapeutic outcomes. Accumulating evidence has also suggested that CAR may be a potential drug target for metabolic disorders and liver cancer by modulating cell cycle progression, energy homeostasis, and cell proliferation. Therefore, identification of CAR activators is of potential importance in both drug development and clinical practice. Notably, while CAR is localized in the nucleus and constitutively activated in immortalized cell lines, it is sequestered in the cytoplasm and translocates to the nucleus upon chemical-provoked activation in primary cultured hepatocytes. Here, we have developed a methodology that takes advantage of nuclear translocation being the first and essential step of CAR activation in human primary hepatocytes to perform high-content screening of human CAR modulators by adapting the EYFP-hCAR translocation assay to a 96-well format with automated sample dispensing and fluorescence imaging analysis.