Abstract
The human pregnane X receptor (hPXR) regulates the expression of critical drug metabolism enzymes. One of such enzymes, cytochrome P450 3A4 (CYP3A4), plays critical roles in drug metabolism in hepatocytes that are either quiescent or passing through the cell cycle. It has been well established that the expression of P450, such as CYP3A4, is markedly reduced during liver development or regeneration. Numerous studies have implicated cellular signaling pathways in modulating the functions of nuclear receptors, including hPXR. Here we report that inhibition of cyclin-dependent kinases (Cdks) by kenpaullone and roscovitine (two small molecule inhibitors of Cdks that we identified in a screen for compounds that activate hPXR) leads to activation of hPXR-mediated CYP3A4 gene expression in HepG2 human liver carcinoma cells. Consistent with this finding, activation of Cdk2 attenuates the activation of CYP3A4 gene expression. In vitro kinase assays revealed that Cdk2 directly phosphorylates hPXR. A phosphomimetic mutation of a putative Cdk phosphorylation site, Ser(350), significantly impairs the function of hPXR, whereas a phosphorylation-deficient mutation confers resistance to Cdk2. Using HepG2 that has been stably transfected with hPXR and the CYP3A4-luciferase reporter, enriched in different phases of the cell cycle, we found that hPXR-mediated CYP3A4 expression is greatly reduced in the S phase. Our results indicate for the first time that Cdk2 negatively regulates the activity of hPXR, and suggest an important role for Cdk2 in regulating hPXR activity and CYP3A4 expression in hepatocytes passing through the cell cycle, such as those in fetal or regenerating adult liver.
Highlights
Nicotinic acetylcholine receptors2 are ligand-gated ion channels expressed mainly in the nervous system and at the neuromuscular junction [1,2,3], they are expressed elsewhere [4]
The nearly identical ligand affinities of ␣4M1/2M1 Nicotinic acetylcholine receptors (nAChRs), the predominant species for ␣42 nAChRs, and the assembly of ␣4M1 and 2M1 subunits into multimers that likely are pentamers suggest that extracellular domains with the M1 domains of ␣4 and 2 subunits form high fidelity structural models of ␣42 nAChRs
The values of dissociation constants for binding between ␣42 nAChRs and [3H]epibatidine, cytisine, nicotine, and acetylcholine are similar to values reported from other laboratories (48 –51, 65– 67)
Summary
Design of Subunit DNA Plasmids—The cDNAs for human ␣4 subunit [37] and human 2 subunit [38] were cloned into pSP64 poly(A) (Promega). The microwells were incubated overnight at 4 °C to allow binding of epitope-tagged subunits and washed with ice-cold buffer B. Values for Kdco and Kdco were estimated by least squares fitting of the equation RE ϩ 2 ϫ RE2 (total concentration of bound [3H]epibatidine) simultaneously to all data sets of [3H]epibatidine binding, where RE and RE2 are defined by Equations 3 and 4. Values for Kdin and Kdin were estimated by least squares fitting of the equation R [1]E ϩ R [2]E (total concentration of bound [3H]epibatidine) from Equations 6 and 7 simultaneously to all relevant data sets. Equation 9 describes the concentration of the [3H]epibatidine-binding species for the one-site competitive inhibition model, Bound3Hepibatidine ϭ Rt/͑1 ϩIt/IC50͒nϩ C (Eq 12). Relative yield helped control for variations in protein expression that depended on time of year or the particular frog from which oocytes were harvested
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