Zangar R C, Bollinger N, Verma S, Karin N J, Lu Y
Cell Biology and Biochemistry, Pacific Northwest National Laboratory, Richland, Washington, USA.
Mol Pharmacol. 2008 Jun;73(6):1652-8. doi: 10.1124/mol.107.043976. Epub 2008 Mar 11.
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.
我们之前观察到，在原代培养的肝细胞中，蛋白酶体的抑制会抑制CYP3A4蛋白水平。由于该结果与如果CYP3A4被蛋白酶体降解的预期结果相反，似乎可能存在另一种易受蛋白酶体降解影响的蛋白质调节CYP3A4表达。在本研究中，我们评估了核因子-κB（NF-κB）途径是否参与该过程。我们的模型系统使用腺病毒系统在源自人癌细胞的HepG2细胞中表达CYP3A4蛋白。与原代肝细胞中的结果相似，用N-苯甲酰氧基羰基（Z）-亮氨酰-亮氨酰-亮氨酸（MG132）抑制蛋白酶体会抑制CYP3A4蛋白水平。我们还发现MG132处理对NF-κB途径有广泛影响，包括下调NF-κB DNA结合活性和IκB激酶（IKK）α水平以及上调IKKβ和抑制性κB水平。用几种结构不同的NF-κB抑制剂处理HepG2细胞也会抑制CYP3A4蛋白水平。当用蛋白质合成的通用抑制剂环己酰亚胺处理HepG2细胞时，与蛋白酶体或NF-κB抑制剂共同处理会加速CYP3A蛋白的丢失。这些结果表明NF-κB活性调节CYP3A4蛋白稳定性，并且提示NF-κB途径是蛋白酶体抑制导致CYP3A4蛋白水平降低的原因。
