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-κB (NF-κB) 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-κB pathway, including down-regulation of NF-κB DNA binding activity and IκB kinase (IKK)α levels and up-regulation of IKKβ and inhibitory κB levels. Treatment of the HepG2 cells with several structurally distinct NF-κB 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-κB inhibitors. These results indicate that NF-κB activity regulated CYP3A4 protein stability, and they suggest that the NF-κB pathway was responsible for the decrease in CYP3A4 protein levels that resulted from the proteasomal inhibition.
Footnotes
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This work was supported by National Institutes of Health grant CA117378 and by the Environmental Biomarkers Initiative, which is a Laboratory Directed Research and Development Program at Pacific Northwest National Laboratory (PNNL). PNNL is operated by Battelle for the U.S. Department of Energy.
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ABBREVIATIONS: P450, cytochrome P450; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; NF-κB, nuclear factor-κB; IκB, inhibitory κB; IKK, IκB kinase; NAI, nuclear factor-κB activation inhibitor; CAPE, caffeic acid phenethyl ester; MPPB, 4-methyl-N1-(3-phenylpropyl)benzene-1,2-diamine; PBS, phosphate-buffered saline; Ad3A4, CYP3A4-expressing adenovirus; AdRed, cytochrome P450 reductase-expressing adenovirus; MOI, multiplicity of infection; ER, endoplasmic reticulum; DiOC6(3), 3,3′-dihexyloxacarbocyanine iodide; RT-PCR, reverse transcription-polymerase chain reaction; RSV, Rous sarcoma virus; CON, control.
- Received November 29, 2007.
- Accepted March 11, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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