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 downregulated 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 siRNAmediated 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 phosphorylationdeficient 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.

Mg2+/Mn2+-Dependent Phosphatase 1A Is Involved in Regulating Pregnane X Receptor–Mediated Cytochrome p450 3A4 Gene Expression

“…3, B and C). These results confirm that PPM1A levels, similar to hPXR activity (Lin et al, 2008;Sivertsson et al, 2013), are regulated in a proliferation-dependent (A) COS-7 cells were cotransfected with FLAGhPXR and PPM1A or FLAG-pcDNA. Twenty-four hours post-transfection, the cells were treated with either DMSO or 10 mM rifampicin for 24 hours and lysed with Triton lysis buffer.…”

“…It is known that hPXR-activated CYP3A4 expression in hepatocytes is regulated in a proliferation-dependent manner (Lin et al, 2008;Sivertsson et al, 2013). It is also known that PPM family phosphatases, including PPM1A, regulate cell growth (Klumpp et al, 2006;Tamura et al, 2006;Yang et al, 2011;Shohat et al, 2012).…”

“…*P , 0.05, determined by unpaired Student's t test. TNFa (Gu et al, 2006;Zhou et al, 2006) and CDK2 (Lin et al, 2008;Sivertsson et al, 2013;Elias et al, 2014) signaling pathways, which are upregulated and essential for hepatocyte proliferation in regenerating livers, inhibit PXR-activated CYP3A expression. Whereas CDK2 phosphorylates and inhibits PXR activity (Lin et al, 2008;Elias et al, 2014), TNFa induces NF-kB activation to inhibit PXRretinoid X receptor (RXRa) interaction (Gu et al, 2006;Xie and Tian, 2006).…”

“…For instance, p53, FOXO1, and nuclear factor-kB (NF-kB) interact with and regulate PXR function (Kodama et al, 2004;Gu et al, 2006;Xie and Tian, 2006;Elias et al, 2013;Kodama and Negishi, 2013). Similarly, posttranslational modifications, including phosphorylation, ubiquitination, acetylation, and sumoylation, regulate PXR activity (Staudinger et al, 2011;Sugatani et al, 2012Sugatani et al, , 2014Sivertsson et al, 2013;Smutny et al, 2013). In particular, kinases such as protein kinase A, protein kinase C, cyclin-dependent kinase 2 (CDK2), p70 S6 kinase, and Ca 2+ /calmodulin-dependent protein kinase II were found to modulate the activity of PXR (Staudinger et al, 2011;Elias et al, 2014;Sugatani et al, 2014).…”

CYP3A4 induction mechanism of polycyclic aromatic hydrocarbons differs from that of rifampicin in PXR binding element

“…There are some reports of new activation pathways of CYP3A4 transcription. For example, cyclin-dependent kinase2 negatively regulates the activity of PXR through phosphorylation of Ser 350 (Lin et al, 2008) and cell proliferation in Huh7 cells (Sivertsson et al, 2013), but the mechanism of CYP3A4 induction by DBA remains unclear. Therefore, further studies are needed to determine the CYP3A4 induction mechanism by DBA.…”