As the major organ for drug detoxification and excretion, the liver is endowed with the capability of inducing drug metabolizing enzymes and transporters in response to drug exposures. Upon activation by drugs such as phenobarbital (PB) and phenytoin, constitutive active/adrostane receptor (CAR) translocates from the cytoplasm to the nucleus in which the receptor forms a heterodimer with retinoid X receptor (RXR) to up-regulate transcription of the genes that encode drug metabolizing enzymes such as cytochromes P450 and the drug transporters [1
]. The function of CAR has now been extended to the regulation of drug-induced repression of hepatic gluconeogenesis, by cross talking with the insulin response FoxO1 transcription factor to repress the genes such as glucose-6-phosphatase
and phosphoenoylpyruvate carboxykinase 1
]. In liver during regeneration, CAR up-regulates the expression of the deiodinase 1
gene increasing thyroid hormone activity [8
]. Also CAR is characterized as the essential factor for PB promotion of development of hepatocellular carcinoma [9
]. Thus, deciphering the molecular mechanism of CAR activation by drugs is now critical for us to understand the receptor-mediated drug effects on liver functions and diseases.
Drug activation of CAR begins with the nuclear translocation from the cytoplasm of liver cells into the nucleus [10
]. Unlike the nuclear steroid hormone receptors, for which their nuclear translocation is dictated by direct ligand binding, CAR is unique in which its nuclear translocation occurs without direct binding of its activators [11
]. Although a cellular signal pathway may be present to retain CAR in the cytoplasm, no such a signal has yet been identified. epithelial cell-transforming gene 2 (ECT2) was originally cloned from epithelial cells and was characterized as a guanine nucleotide exchange factor bearing oncogenic activity [12
]. We have linked ECT2 with CAR based on observations obtained in two independent analyses: (1) cDNA microarray analysis of wildtype and Car-/-
mice after hepatectomy and PB treatment; and (2) looking for genes up-regulated during PB-promoted development of liver tumors [9
]. Given these serendipitous findings, we pursued more detailed studies of ECT2, examining whether or not it regulates drug-induced nuclear translocation of CAR. By tail vein injection of expression plasmid DNAs, fluorescent protein tagged-ECT2 and its deletion mutants were directly co-expressed with CAR in mouse livers of Car-/-
mice. ECT2 and CAR were also co-expressed in HepG2 cells for co-immunoprecipitation assays to define the molecular basis for their interaction. Thus, here we present experimental evidence that ECT2 directly interacts with CAR in the cytoplasm of liver cells to repress PB-induced CAR nuclear translocation.