The nuclear factor-kappa B pathway regulates cytochrome P450 3A4 protein stability

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-kappaB (NF-kappaB) 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-kappaB pathway, including down-regulation of NF-kappaB DNA binding activity and IkappaB kinase (IKK)alpha levels and up-regulation of IKKbeta and inhibitory kappaB levels. Treatment of the HepG2 cells with several structurally distinct NF-kappaB 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-kappaB inhibitors. These results indicate that NF-kappaB activity regulated CYP3A4 protein stability, and they suggest that the NF-kappaB pathway was responsible for the decrease in CYP3A4 protein levels that resulted from the proteasomal inhibition.