Received for publication June 13, 2008.
Revised June 8, 2009.
Accepted for publication June 9, 2009.

Thyroid Hormone Is Necessary for Expression of Constitutive Androstane Receptor in Rat Hepatocytes

Abstract

Small hepatocytes are hepatocyte progenitor cells that possess the capability of maturation and cryopreservation. When cryopreserved rat small hepatocytes were cultured in serum-free medium, the protein expression and the inducibility of CYP 1A1/2, CYP2E1, and CYP3A were maintained but those of CYP2B1 were lost. In this study we investigated the cause of the loss of CYP2B1 expression in cryopreserved small hepatocytes by RT-PCR, immunoblotting, and chromatin-immunoprecipitation assay. Expression of mRNA and protein of the nuclear receptor, constitutive androstane receptor (CAR), which regulates the expression of CYP2B1, was inhibited in the serum-free culture of cryopreserved small hepatocytes, whereas they were expressed in that of subcultured small hepatocytes. Serum application dramatically induced CAR expression in the culture of cryopreserved small hepatocytes. The addition of very low concentrations of thyroid hormones (THs; T₃, 5x10^-12 M; T₄, 5x10^-12~5x10^-10 M) to the medium also induced the expression of CAR and CYP2B1. Moreover, CYP2B1 expression was induced by administration of phenobarbital. In rats with hypothyroidism induced by thyroidectomy and 6-propyl-2-thiouracil treatment, the expression of CAR and CYP2B1 was strongly repressed. Although THs do not directly regulate the expression of CAR, they may be important for rat hepatocytes to regulate CYP2B1 through CAR expression in the physiological condition.

Key words: CAR, CYP gene regulation, CYP2B, gene regulation, hepatocytes, in vitro toxicity assays