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

  1. Poulomi Acharya,
  2. Juan C. Engel and
  3. Maria Almira Correia
  1. Departments of Cellular & Molecular Pharmacology (P.A., M.A.C.), Pharmaceutical Chemistry (M.A.C.), Biopharmaceutical Sciences (M.A.C.), and Pathology (J.C.E.), the Liver Center (P.A., M.A.C.), and Sandler Center for Basic Research in Parasitic Diseases (J.C.E.), University of California, San Francisco, California
  1. Address correspondence to:
    M. A. Correia, Dept. of Cellular and Molecular Pharmacology, Mission Bay Campus, Genentech Hall, 600 16th Street, N572F/Box 2280, University of California, San Francisco, CA 94158. E-mail: almira.correia{at}ucsf.edu

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

  • ↵1 CYP3A or P450s 3A refer to rat liver P450s 3A2, 3A23, 3A18, and 3A9 and/or human liver CYP3A4/CYP3A5.

  • ↵2 Higher concentrations of MG132 were used in these hepatocyte incubations; as a peptide aldehyde, it can be readily quenched by the relatively high GSH concentrations present in these freshly isolated cells.

  • ↵3 This CYP3A at the plasma membrane was found oriented intracellularly toward the cytosol, rather than extracellularly, as documented for several other P450s (Loeper et al., 1993; Neve and Ingelman-Sundberg, 2000).

  • ↵4 In this context, it is worth noting on the one hand that proteasomal inhibitor-induced eIF2α phosphorylation is the initial event that results in the suppression of protein translation and, on the other, apoptosis via delayed translational reinitiation at upstream open reading frames results in the enhanced expression of apoptotic bZip regulators such as ATF4, CHOP, and ATF3 (Harding et al., 2002; Jiang et al., 2004; Jiang and Wek, 2005a). Thus, CYP3A suppression and apoptosis are two distinct manifestations of MG132-induced eIF2α phosphorylation. This is underscored by the much lower MG132 concentrations required to suppress CYP3A synthesis (Fig. 6C) than those required to induce DNA fragmentation (Fig. 10C).

  • ↵5 Our results, however, do not fully explain the 40% inhibition of protein synthesis observed at a 5 μM MG132 concentration in primary rat hepatocytes (Noreault-Conti et al., 2006). It is unclear whether this discrepancy is due to rat strain differences (i.e., Fisher 344 strain versus Sprague-Dawley strain) or to the fact that in that study, the hepatocytes were plated on Matrigel with no overlay, whereas ours were plated on collagen-coated Permanox plates with a Matrigel overlay. Such hepatocyte sandwich cultures would significantly protect cells from stresses, including oxidative stress due to direct exposure to 95% air. It is conceivable that differences in stress predisposition and/or relative ER stress levels may explain this discrepancy. It is noteworthy, however, that in certain cells, as little as 0.1 μM MG132 can activate UPR and PERK, resulting in eIF2α phosphorylation and consequent inhibition of de novo protein synthesis (Nishitoh et al., 2002; Jiang and Wek, 2005a).

  • ↵6 Double-stranded RNA-activated PKR is usually induced by viral inducers or class 1 interferons that were not specifically included in the cell culture; it therefore was not examined.

  • 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.

  • ↵Graphic The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.

    • Accepted June 11, 2009.
    • Received March 9, 2009.
« Previous | Next Article »Table of Contents

This Article

  1. Published online before print June 11, 2009, doi: 10.1124/mol.109.056002 Molecular Pharmacology September 2009 vol. 76 no. 3 503-515
  1. » Abstract
  2. Full Text
  3. Full Text (PDF)
  4. Data Supplement
  5. All Versions of this Article:
    1. mol.109.056002v1
    2. 76/3/503 most recent

Classifications

Responses

  1. Submit a response
  2. No responses published

Current Issue

  1. November 2009, 76 (5)
  1. Current Issue
  1. Alert me to new issues of Molecular Pharmacology