A phosphorylation-deficient mutation (S350A) in the PXR protein significantly induced the CYP3A4 transcription. Moreover, the CDK inhibitor roscovitine stimulated the expression of CYP3A4. Knock-down of CDK2 in proliferating control cells increased the CYP3A4 and PXR protein levels.

Trypsinization of the confluent cells and replating them subconfluent resulted in a decrease in CYP3A4 and PXR expression back to levels observed in subconfluent control cells, whereas the CDK2 levels increased. Cyclin-dependent kinase 2 (CDK2), a known modulator of the cell cycle and a negative regulator of PXR, was more highly expressed in proliferating control cells. The PXR ligand rifampicin further increased the expression of CYP3A4, and siRNA-mediated knock-down of PXR in confluent cells resulted in decreased CYP3A4 expression. PXR expression was increased, and PXR protein accumulated in the nuclei during confluent growth. Reporter analysis revealed that the CYP3A4 gene was transcriptionally activated during confluence in a process involving pregnane X receptor (PXR). Here, the mechanisms behind were investigated, and a transcriptome analysis revealed significant up-regulation of liver-specific functions, whereas pathways related to proliferation and cell cycle were down-regulated in the confluent cells. We have previously shown that confluent growth of the human hepatoma cell line Huh7 substantially induces the CYP3A4 mRNA, protein, and activity levels.

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