Indirect activation of constitutive androstane receptor in three-dimensionally cultured HepG2 cells.

Constitutive androstane receptor (CAR), a member of the nuclear receptor superfamily, is retained as an inactive form phosphorylated at threonine in the cytoplasm of hepatocytes. Upon activation, CAR is dephosphorylated to move into the nucleus and induces the transcription of genes. Thus, nuclear translocation is a key step for CAR activation in hepatocytes. However, this nuclear translocation has not been demonstrated in conventional two-dimensionally-cultured immortalized cell lines such as HepG2, in which CAR spontaneously accumulates in the nucleus. In this study, we showed that treatment with the indirect CAR activator phenobarbital activated transcription of the CYP3A4 gene in three-dimensionally (3D)-cultured HepG2 cells. CAR was retained as its phosphorylated form in the cytoplasm and was translocated to the nucleus in 3D-cultured HepG2 cells in response to treatment with phenobarbital. Moreover, okadaic acid and epidermal growth factor, were found to repress phenobarbital-induced CAR nuclear translocation and subsequent activation of the CYP3A4 gene promoter. These results suggested that 3D-cultured HepG2 cells properly regulated CAR activation as has been observed in hepatocytes.

Intracellular localization of pregnane X receptor in HepG2 cells cultured by the hanging drop method.

Pregnane X receptor (PXR) is localized in the cytoplasm of liver cells, whereas it is localized in the nucleus of monolayer-cultured HepG2 cells. Since cultured cells are affected by the microenvironment in which they are grown, we studied the effect of three-dimensional (3D) culture on the localization of PXR in HepG2 cells using the hanging drop method. The results showed that PXR was retained in the cytoplasm of HepG2 cells and other human hepatocarcinoma cell lines (FLC5, FLC7 and Huh7) when they were cultured by the hanging drop method. Treatment with rifampicin, a ligand of PXR, translocated PXR from the cytoplasm to nucleus and increased expression levels of CYP3A4 mRNA in HepG2 cells cultured by the hanging drop method. These findings suggest that 3D culture is a key factor determining the intracellular localization of PXR in human hepatocarcinoma cells and that PXR that becomes retained in the cytoplasm of HepG2 cells with 3D culture has functions of nuclear translocation and regulation of target genes in response to human PXR ligands. Three-dimensionally cultured hepatocarcinoma cells would be a useful tool to evaluate induction potency of drug candidates and also to study mechanisms of nuclear translocation of PXR by human PXR ligands.