In the regulation of cytochrome P450 genes, phenobarbital targets LKB1 for necessary activation of AMP-activated protein kinase

Abstract

Transcriptional activation of cytochrome P450 (CYP) genes and various drug metabolizing enzymes by the prototypical inducer phenobarbital (PB) and many other drugs and chemicals is an adaptive response of the organism to exposure to xenobiotics. The response to PB is mediated by the nuclear receptor constitutive androstane receptor (CAR), whereas the chicken xenobiotic receptor (CXR) has been characterized as the PB mediator in chicken hepatocytes. Our previous results suggested an involvement of AMP-activated protein kinase (AMPK) in the molecular mechanism of PB induction. Here, we show that the mechanism of AMPK activation is related to an effect of PB-type inducers on mitochondrial function with consequent formation of reactive oxygen species (ROS) and phosphorylation of AMPK by the upstream kinase LKB1. Gain- and loss-of-function experiments demonstrate that LKB1-activated AMPK is necessary in the mechanism of drug induction and that this is an evolutionary conserved pathway for detoxification of exogenous and endogenous chemicals. The activation of LKB1 adds a proximal target to the so far elusive sequence of events by which PB and other drugs induce the transcription of multiple genes.

Fig. 1.

Chicken AMPKα subunits are activated by PB-type inducers. (A) LMH cells were treated with increasing doses of PB or metyrapone for 1h. AMPK activity is shown as percentage of the control. ∗, P < 0.01; ∗∗, P < 0.05. (B) Phosphorylation of AMPK-Thr-172 and ACC-Ser-79 is shown by Western blot after 1 h treatment with 500 μM PB or metyrapone (M).

Fig. 2.

Activation or overexpression of AMPKα subunits affect CYP2H1, CYP3A37, and ALAS1 gene expression. (A) LMH cells were treated with 1 mM AICAR (AIC) or metformin (Met), 0.2 mM DNP, 1 mM NaN₃, or 1 μM rotenone (Rot) for 16 h. Gene expression was analyzed by RT-PCR. (B) LMH cells transiently transfected with AMPKα subunits were treated with 500 μM PB or metyrapone (M) for 16 h. Gene expression was analyzed by RT-PCR. ∗, P < 0.01; ∗∗, P < 0.05.

Fig. 3.

Down-regulation of AMPKα activity by diced siRNA or Compound C decreases PB- and metyrapone-mediated induction of CYP2H1, CYP3A37, and ALAS1. (A) LMH cells were transiently transfected with AMPKα-specific siRNA. AMPKα protein expression was detected by Western blot. (B) LMH cells transiently transfected with siRNA were treated with 500 μM PB or metyrapone for 16 h. mRNA expression was measured by RT-PCR. ∗, P < 0.01. (C) Activation of AMPK by 500 μM PB or metyrapone with or without 30-min pretreatment with 20 μM Compound C is shown by Western blot evidencing the phosphorylation of AMPK-Thr-172 and ACC-Ser-79. (D) LMH cells were incubated 16 h with 500 μM PB or metyrapone with or without pretreatment for 30 min by 20 μM Compound C. mRNA levels were measured by RT-PCR. ∗, P < 0.01. M, metyrapone.

Fig. 4.

PB and metyrapone trigger the interaction of AMPKα with the upstream kinase LKB1 and affect mitochondrial membrane potential, ROS production, and LKB1 phosphorylation. (A) Immunoprecipitation of overexpressed HA-LKB1 by anti-HA antibody upon 500 μM PB or metyrapone. (B) Immunoprecipitation of overexpressed Myc-AMPKα upon 500 μM PB or metyrapone treatment. (C) JC-1 fluorescence was detected after 1 h treatment with 500 μM PB or metyrapone, 0.4 mM DNP, or 1 mM AICAR. Results are expressed as the 590 nm/540 nm fluorescence ratio in comparison with the control sample ratio. P < 0.05. (D) DCF fluorescence was detected after 1-h treatment with 500 μM PB or metyrapone, 5 μM rotenone, or 1 mM AICAR for 1 h. The result is shown as fold increase in comparison with the control sample. P < 0.01. (E) Phosphorylation of LKB1-Ser-428 upon PB and metyrapone is proven in a Western blot. M, metyrapone; Rot, rotenone; AIC, AICAR.

Fig. 5.

Decrease in intracellular ROS production by UCP-1 overexpression or by NAC-mediated scavenging attenuates drug-mediated increase of CYP2H1, CYP3A37 and ALAS1 gene expression and AMPK activity. (A) LMH cells transiently transfected with UCP-1 were incubated with 500 μM PB or metyrapone, or 1 mM AICAR for 16 h. Gene expression was detected by RT-PCR. ∗, P < 0.01. (B) LMH cells were incubated with 500 μM PB or metyrapone, or 1 mM AICAR, with or without 10 mM NAC for 16 h. mRNA levels were measured by RT-PCR. ∗, P < 0.01. (C) LMH WT or LMHρ⁰ cells were incubated with 500 μM PB or metyrapone for 16 h. mRNA levels were detected by RT-PCR. ∗, P < 0.01. (D) Activation of AMPK after 1 h treatment with 500 μM PB or metyrapone, or 1 mM AICAR is shown by Western blot evidencing the phosphorylation of AMPK-Thr-172 and ACC-Ser-79. M, metyrapone, AIC, AICAR.