Hepatic CYP3A protein suppression by proteasomal inhibitors is due to induction of ER‐stress, activation of the eIF2‐alpha kinase PERK leading to translational arrest

Abstract

Hepatic cytochromes P450 3A (CYP3A) 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 MG132 and MG262 stabilize CYP3A proteins. However, more recently MG132 was reported to suppress CYP3A due to an NF?B‐mediated regulation of protein stability. Because 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 5‐10 µM with marked suppression at >100 µM. Because such CYP3A suppression could be due to MG132‐induced ER‐stress, we monitored the activity of PERK, the ER‐stress‐activated eIF2α kinase. Indeed, we found a significant (5‐fold) MG132 concentration‐dependent PERK autophosphorylation, along with a 8‐fold increase in eIF2α‐phosphorylation. Pulse‐chase and CYP3A immunoprecipitation analyses documented the dramatic translational arrest of total hepatic protein as well as CYP3A protein syntheses; thereby confirming that at high concentrations, MG132 is indeed cytotoxic and can suppress CYP3A synthesis.Supported by NIH grants GM44037, DK26506 and DK26743.