Molecular basis for rhythmic expression of CYP3A4 in serum-shocked HepG2 cells.
OBJECTIVE
Although the pharmacokinetics of several drugs that are mainly eliminated by the CYP3A4 metabolism vary according to their dosing time, the mechanism of the variation remains poorly understood. In this study, we investigated how the 24-h oscillation in the expression of CYP3A4 mRNA was generated in hepatic cells.


RESULTS
As brief exposure of HepG2 cells to 50% serum induced the 24-h oscillation in the expression of clock genes, serum-shocked HepG2 cells were employed as an in-vitro model to study the molecular mechanism underlying the circadian clock in the human liver. Both mRNA levels and metabolic activity of CYP3A4 in serum-shocked HepG2 cells fluctuated rhythmically with a period length of about 24 h. The oscillation in the expression of the CYP3A4 gene seemed to be the underlying cause of the rhythmic change in its metabolic activity. Luciferase reporter gene analysis and electrophoretic mobility shift assay revealed that the circadian transcriptional factor, D-site-binding protein (DBP), activated the transcription of the CYP3A4 gene by binding to the DNA sequence near the upstream of the transcriptional start site. The transactivation of the CYP3A4 gene by DBP was repressed by the E4 promoter-binding protein-4 (E4BP4), a negative component of the circadian clock.


CONCLUSIONS
Results from this study suggest that DBP and E4BP4 might consist of a reciprocating mechanism in which DBP activates the transcription of the CYP3A4 gene during the time of day when DBP is abundant, and E4BP4 suppresses the transcription at other times of day. Our current findings provide a molecular link between the circadian clock and the xenobiotic metabolism.
Takiguchi
Takako
T
Pharmaceutics, Division of Clinical Pharmacy, Department of Medico-Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Tomita
Miho
M
Matsunaga
Naoya
N
Nakagawa
Hiroo
H
Koyanagi
Satoru
S
Ohdo
Shigehiro
S
D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors / Q000378:metabolism; D002460:Cell Line; D002940:Circadian Rhythm / Q000235:genetics* / Q000502:physiology*; D003470:Culture Media; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010597:Pharmacogenetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation
D050976:Basic-Leucine Zipper Transcription Factors; D003470:Culture Media; C063898:DBP protein, human; D004268:DNA-Binding Proteins; C497440:NFIL3 protein, human; D012333:RNA, Messenger; D014157:Transcription Factors; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human