Abstract
Hepatic cytochromes P450 3A (P450s 3A) are endoplasmic reticulum (ER)-proteins, responsible for xenobiotic metabolism. They are degraded by the ubiquitin-dependent 26S proteasome. Consistent with this, we have shown that proteasomal inhibitors N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) and N-benzoyloxycarbonyl-Leu-Leu-Leu-B(OH)2 (MG262) stabilize CYP3A proteins. However, MG132 has been reported to suppress P450s 3A as a result of impaired nuclear factor-κB activation and consequently reduced CYP3A protein stability. Because the MG132 concentration used in those studies was 10-fold higher than that required for CYP3A stabilization, we examined the effect of MG132 (0-300 μM) concentration-dependent proteasomal inhibition on CYP3A turnover in cultured primary rat hepatocytes. We found a biphasic MG132 concentration effect on CYP3A turnover: Stabilization at 5 to 10 μM with marked suppression at >100 μM. Proteasomal inhibitors reportedly induce ER stress, heat shock, and apoptotic response. At these high MG132 concentrations, such CYP3A suppression could be due to ER stress induction, so we monitored the activity of PERK [PKR (RNA-dependent protein kinase)-like ER kinase (EIF2AK3)], the ER stress-activated eukaryotic initiation factor 2α (eIF2α) kinase. Indeed, we found a marked (≈4-fold) MG132 concentration-dependent PERK autophosphorylation, along with an 8-fold increase in eIF2α-phosphorylation. In parallel, MG132 also activated GCN2 [general control nonderepressible-2 (EIF2AK4)] eIF2α kinase in a concentration-dependent manner, but not the heme-regulated inhibitor eIF2α kinase [(EIF2AK1)]. Pulse-chase, immunoprecipitation/immunoblotting analyses documented the consequently dramatic translational shutoff of total hepatic protein, including but not limited to CYP3A and tryptophan 2,3-dioxygenase protein syntheses. These findings reveal that at high concentrations, MG132 is indeed cytotoxic and can suppress CYP3A synthesis, a result confirmed by confocal immunofluorescence analyses of MG132-treated hepatocytes.
Footnotes
- Received March 9, 2009.
- Accepted June 11, 2009.
This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK26506] and the National Institutes of Health National Institute of General Medicine [Grant GM44037]. We also acknowledge the UCSF Liver Center Core on Cell and Tissue Biology supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant P30-DK26743].
ABBREVIATIONS: P450, cytochrome P450; ER, endoplasmic reticulum; DDI, drug-drug interaction; Ub, ubiquitin; UPD, ubiquitin-mediated 26S proteasomal degradation; ALD, autophagic-lysosomal degradation; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; MG262, N-benzoyloxycarbonyl-Leu-Leu-Leu-B(OH)2; ERAD, ER-associated degradation; NFκB, nuclear factor κB; IκB, inhibitor of nuclear factor κB; UPR, unfolded protein response; PERK, PKR-like ER-bound eIF2α-kinase; PKR, RNA-dependent protein kinase; eIF2α, α-subunit of the eukaryotic initiation factor 2; GCN2, general control nonderepressible-2; WME, William's medium E; BSA, bovine serum albumin; UCSF, University of California San Francisco; PMSF, phenylmethylsulfonyl fluoride; Dex, dexamethasone; HRI, Heme-regulated inhibitor; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; DAPI, 4,6-diamidino-2-phenylindole; AK, adenylate kinase; TTBS, Tris-buffered saline-Tween 20; TDO, tryptophan 2,3-dioxygenase; PBS, phosphate-buffered saline; HMM, high molecular mass; MEF, mouse embryonic fibroblast; eIF2αP, phosphorylated eIF2α; RHL, rat hepatocyte lysate.
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RETRACTION: Hepatic CYP3A Suppression by High Concentrations of Proteasomal Inhibitors: A Consequence of Endoplasmic Reticulum (ER) Stress Induction, Activation of RNA-Dependent Protein Kinase-Like ER-Bound Eukaryotic Initiation Factor 2α (eIF2α)-Kinase (PERK) and General Control Nonderepressible-2 eIF2α Kinase (GCN2), and Global Translational Shutoff
