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 CYP1A1/2, 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 reverse transcription-polymerase chain reaction, 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; 3,5,3′-triiodothyronine, 5 × 10–12 M; thyroxine, 5 × 10–12-5 × 10–10 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.
Footnotes
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This work was supported in part by the Ministry of Education, Culture, Sports, Science, and Technology, Japan [Grant 17390353]; the Ministry of Health, Labour, and Welfare; Health and Labour Sciences Research Grants; Research on Advanced Medical Technology; and the Suhara Memorial Foundation.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.022905.
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ABBREVIATIONS: P450, cytochrome P450; SH, small hepatocyte; AhR, aryl hydrocarbon receptor; CAR, constitutive androstane receptor; RXR, retinoid X receptor; PXR, pregnane X receptor; PB, phenobarbital; cryo-SH, cryopreserved small hepatocyte; TH, thyroid hormone; MH, mature hepatocyte; PTU, 6-propyl-2-thiouracil; T3, 3,5,3′-triiodothyronine; T4, thyroxine; FBS, fetal bovine serum; PBS, phosphate-buffered saline; G3PDH, glycerol 3 phosphate dehydrogenase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; ChIP, chromatin immunoprecipitation; TR, thyroid hormone receptor; sub-SH, subcultured small hepatocyte; TRE, TH-responsive element; RT-PCR, reverse transcription-polymerase chain reaction; Dio1, 5′-deiodinase type 1; ME1, malic enzyme 1; Thrsp, thyroid hormone-responsive gene.
- Accepted June 9, 2009.
- Received June 13, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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