A physiological role of AMP-activated protein kinase in phenobarbital-mediated constitutive androstane receptor activation and CYP2B induction.
CAR (constitutive androstane receptor) is a nuclear receptor that regulates the transcription of target genes, including CYP (cytochrome P450) 2B and 3A. The transactivation by CAR is regulated by its subcellular localization; however, the mechanism that governs nuclear translocation has yet to be clarified. It has been reported recently that AMPK (AMP-activated protein kinase) is involved in phenobarbital-mediated CYP2B induction in a particular culture system. We therefore investigated in vivo whether AMPK is involved in the activation of CAR-dependent gene expression. Immunoblot analysis using an antibody which recognizes Thr-172-phosphorylated AMPKalpha1/2 revealed phenobarbital-induced AMPK activation in rat and mouse livers as well. Phenobarbital, however, failed to increase the liver phospho-AMPK level of tumour-bearing rats in which CAR nuclear translocation had been impaired. In in vivo reporter gene assays employing PBREM (phenobarbital-responsive enhancer module) from CYP2B1, an AMPK inhibitor 8-bromo-AMP abolished phenobarbital-induced transactivation. In addition, Cyp2b10 gene expression was attenuated by 8-bromo-AMP. Forced expression of a dominant-negative mutant and the wild-type of AMPKalpha2 in the mouse liver suppressed and further enhanced phenobarbital-induced PBREM-reporter activity respectively. Moreover, the AMPK activator AICAR (5-amino-4-imidazolecarboxamide riboside) induced PBREM transactivation and an accumulation of CAR in the nuclear fraction of the mouse liver. However, AICAR and metformin, another AMPK activator, failed to induce hepatic CYP2B in mice and rats. These observations suggest that AMPK is at least partly involved in phenobarbital-originated signalling, but the kinase activation by itself is not sufficient for CYP2B induction in vivo.
Shindo
Sawako
S
Department of Biochemical Toxicology, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan.
Numazawa
Satoshi
S
Yoshida
Takemi
T
D055372:AMP-Activated Protein Kinases; D000818:Animals; D000090702:Constitutive Androstane Receptor; D019362:Cytochrome P-450 CYP2B1 / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D009097:Multienzyme Complexes / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D017208:Rats, Wistar; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D000090702:Constitutive Androstane Receptor; D009097:Multienzyme Complexes; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D019362:Cytochrome P-450 CYP2B1; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D010634:Phenobarbital