Laboratory of DDS Design and Drug Disposition, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
Laboratory of DDS Design and Drug Disposition, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
Biochem Pharmacol. 2019 Oct;168:26-37. doi: 10.1016/j.bcp.2019.06.011. Epub 2019 Jun 13.
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.
组成型雄烷受体（CAR）是核受体超家族的一员，在肝细胞的细胞质中以磷酸化的形式保持非活性状态。CAR 被激活后，会去磷酸化并进入细胞核，从而诱导基因转录。因此，核易位是 CAR 在肝细胞中激活的关键步骤。然而，这种核易位尚未在常规的二维培养的永生化细胞系（如 HepG2）中得到证实，在这些细胞系中，CAR 会自发地积累在细胞核中。在这项研究中，我们发现间接 CAR 激活剂苯巴比妥处理能够激活三维培养的 HepG2 细胞中 CYP3A4 基因的转录。苯巴比妥处理后，CAR 以其磷酸化形式保留在细胞质中，并转移到细胞核中。此外，发现岗田酸和表皮生长因子抑制苯巴比妥诱导的 CAR 核易位和随后 CYP3A4 基因启动子的激活。这些结果表明，三维培养的 HepG2 细胞能够像肝细胞一样正确调节 CAR 的激活。
