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Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters
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Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

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CRY1/2 regulate rhythmic CYP2A5 in mouse liver through repression of E4BP4

Luomin Lin1, Yuwei Huang2, Jinyi Wang3

  • 1College of Pharmacy, Jinan University, Guangzhou, China.

Biochemical Pharmacology
|October 5, 2023
PubMed

Insights

Circadian clock proteins CRY1 and CRY2 regulate liver enzyme CYP2A5 expression and activity by inhibiting E4BP4. This finding clarifies mechanisms of drug metabolism rhythms.

Area of Science:

  • Chronobiology
  • Molecular Pharmacology
  • Hepatology

Background:

  • Cytochrome P450 2A5 (CYP2A5) exhibits circadian rhythms in expression and activity, crucial for drug metabolism.
  • The precise molecular mechanisms governing CYP2A5's circadian regulation remain incompletely understood.

Purpose of the Study:

  • To investigate the role of circadian clock proteins CRY1 and CRY2 in the circadian regulation of hepatic CYP2A5.
  • To elucidate the molecular mechanisms by which CRY1/2 influence CYP2A5 expression and activity.

Main Methods:

  • Utilized Cry1-null and Cry2-null mice models for in vivo studies.
  • Employed AML-12, Hepa1-6, and HepG2 cell lines for in vitro validation.
  • Assessed CYP2A5 activity via coumarin 7-hydroxylation, and quantified mRNA/protein levels using qPCR and Western blotting.
  • Investigated regulatory mechanisms using luciferase reporter assays, ChIP, and Co-IP.

Main Results:

  • Ablation of Cry1 or Cry2 in mice led to reduced hepatic CYP2A5 mRNA and protein levels, and abolished diurnal rhythms.
  • CYP2A5 activity was decreased and lost its time-dependency in knockout mice, correlating with increased hepatotoxicity.
  • Cell-based assays confirmed CRY1/2 positively regulate CYP2A5 expression and its circadian rhythm.
  • CRY1/2 were found to interact with E4BP4, thereby repressing E4BP4's inhibitory effect on Cyp2a5 transcription.

Conclusions:

  • CRY1 and CRY2 are key regulators of rhythmic CYP2A5 expression and activity in mouse liver.
  • The mechanism involves CRY1/2 repressing the inhibitory action of E4BP4 on Cyp2a5 transcription.
  • These findings enhance the understanding of circadian control over drug metabolism and pharmacokinetics.
Keywords:
CRY1CRY2CYP2A5ChronopharmacokineticsCircadian rhythm

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