title: Induced CYP3A4 expression in confluent Huh7 hepatoma cells as a result of decreased cell proliferation and subsequent pregnane X receptor activation.
abstract: We have previously shown that confluent growth of the human hepatoma cell line Huh7 substantially induces the CYP3A4 mRNA, protein, and activity levels. 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. Reporter analysis revealed that the CYP3A4 gene was transcriptionally activated during confluence in a process involving pregnane X receptor (PXR). PXR expression was increased, and PXR protein accumulated in the nuclei during confluent growth. 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. 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. 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. Knock-down of CDK2 in proliferating control cells increased the CYP3A4 and PXR protein levels. Moreover, the CDK inhibitor roscovitine stimulated the expression of CYP3A4. A phosphorylation-deficient mutation (S350A) in the PXR protein significantly induced the CYP3A4 transcription. In conclusion, the data strongly suggest that the increased CYP3A4 expression in confluent Huh7 cells is caused by the endogenous induction of PXR as a result of cell-cell contact inhibited proliferation and subsequent decreased CDK2 activities, indicating an endogenous, non-ligand-dependent regulation of PXR and CYP3A4, possibly of physiologic and pharmacological significance.
authors.affiliation: Karolinska Institutet, Department of Physiology and Pharmacology, Nanna Svartz v. 2, SE-171 77 Stockholm, Sweden. louise.sivertsson@ki.se
authors.forename: Louise
authors.initials: L
authors.lastname: Sivertsson
authors.forename: Irene
authors.initials: I
authors.lastname: Edebert
authors.forename: Margareta Porsmyr
authors.initials: MP
authors.lastname: Palmertz
authors.forename: Magnus
authors.initials: M
authors.lastname: Ingelman-Sundberg
authors.forename: Etienne P A
authors.initials: EP
authors.lastname: Neve
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle / Q000235:genetics; D048708:Cell Growth Processes / Q000235:genetics; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics; D006801:Humans; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation / Q000235:genetics; D000077297:Pregnane X Receptor; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000235:genetics; D011987:Receptors, Steroid / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D059467:Transcriptome; D015854:Up-Regulation
chemical_list: D000077297:Pregnane X Receptor; D034741:RNA, Small Interfering; D011987:Receptors, Steroid; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2