The Nuclear Factor-κB Pathway Regulates Cytochrome P450 3A4 Protein Stability

Abstract

We have previously observed that CYP3A4 protein levels are suppressed by inhibition of the proteasome in primary cultured hepatocytes. Because this result is opposite of what would be expected if CYP3A4 were degraded by the proteasome, it seemed likely that there might be another protein susceptible to proteasomal degradation that regulated CYP3A4 expression. In this study, we evaluated whether the nuclear factor-B (NF-B) pathway was involved in that process. Our model system used an adenovirus system to express CYP3A4 protein in HepG2 cells, which are derived from human cancer cells. Similar to results in primary hepatocytes, the inhibition of the proteasome with N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) suppresses CYP3A4 protein levels. We also found that MG132 treatment had a broad affect on the NF-B pathway, including down-regulation of NF-B DNA binding activity and IB kinase (IKK)␣ levels and up-regulation of IKK␤ and inhibitory B levels. Treatment of the HepG2 cells with several structurally distinct NF-B inhibitors also suppressed CYP3A4 protein levels. When the HepG2 cells were treated with cycloheximide, a general inhibitor of protein synthesis, the loss of CYP3A4 protein was accelerated by cotreatment with either proteasome or NF-B inhibitors. These results indicate that NF-B activity regulated CYP3A4 protein stability, and they suggest that the NF-B pathway was responsible for the decrease in CYP3A4 protein levels that resulted from the proteasomal inhibition.

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

“…Our findings described below reveal that MG132 had a biphasic concentration-dependent effect on immunochemically detectable CYP3A levels in cultured rat hepatocytes: stabilization of CYP3A at lower concentrations and a marked suppression at higher concentrations. However, we show that this suppression stems from MG132-induced unfolded protein response (UPR) and consequent ER stress, activation of both PERK [PKR (RNA-dependent protein kinase)-like ER kinase (EIF2AK3)], the resident ER stress-inducible eIF2␣ kinase, and GCN2 [general control nonderepressible-2 (EIF2AK4)] eIF2␣ kinase and consequent global suppression of hepatic protein synthesis, and was not due to reduced CYP3A protein stability as reported previously (Zangar et al, 2008). These findings once again underscore the essential role of UPD in CYP3A ERAD, as well as the concentrations of the proteasomal inhibitors critical for its documentation.…”

“…Although some reports claimed that this suppression is at the mRNA and protein level (Noreault-Conti et al, 2006), others claimed it was due to NFB-mediated regulation of CYP3A protein stability (Zangar et al, 2008). Accordingly, evidence was provided for a reduced NFB activation as a result of MG132-mediated inhibition of proteasomal function (Zangar et al, 2008).…”

“…Accordingly, evidence was provided for a reduced NFB activation as a result of MG132-mediated inhibition of proteasomal function (Zangar et al, 2008). NFB activation requires unleashing from its inhibitory IB regulators via proteasomal degradation.…”

Increased Levels of Fatty Acids Contributed to Induction of Hepatic CYP3A4 Activity Induced by Diabetes — In Vitro Evidence From HepG2 Cell and Fa2N-4 Cell Lines

“…Similarly, both the PI3K and PKC inhibitor also amelio rated the suppressive effect of acetoacetate on CYP2E1 mRNA expression (53). There was a report showing that NFkB plays a pivotal role in the regulation of CYP3A4 through interactions of NFkB with the PXRretinoid X receptor (RXR) complex (54) or the impact on CYP3A4 protein stability (55). Moreover, fatty acid-induced muscle insulin resistance and glucose uptake dysfunction were involved in PKC activation and oxidative stressactivated signaling pathways (56).…”

Metabolite Formation Kinetics and Intrinsic Clearance of Phenacetin, Tolbutamide, Alprazolam, and Midazolam in Adenoviral Cytochrome P450-Transfected HepG2 Cells and Comparison with Hepatocytes and In Vivo

“…By means of this strategy, high levels of functional transgene expression can be achieved without causing major changes in the expression of other constitutive or inducible hepatic genes (Castell et al, 1997). The HepG2 cells transduced with adenoviral constructs have been used to study either the mechanisms involved in the control of the P450 expression or the role of a particular P450 enzyme in the bioactivation of hepatotoxicants (Bai and Cederbaum, 2004;Vignati et al, 2005;Zangar et al, 2008). However, their utility in the prediction of metabolic clearance has not yet been explored.…”