PXR stimulates growth factor-mediated hepatocyte proliferation by cross-talk with the FOXO transcription factor.

Growth factor-mediated hepatocyte proliferation is crucial in liver regeneration and the recovery of liver function after injury. The nuclear receptor, pregnane X receptor (PXR), is a key transcription factor for the xenobiotic-induced expression of genes associated with various liver functions. Recently, we reported that PXR activation stimulates xenobiotic-induced hepatocyte proliferation. In the present study, we investigated whether PXR activation also stimulates growth factor-mediated hepatocyte proliferation. In G0 phase-synchronized, immortalized mouse hepatocytes, serum or epidermal growth factor treatment increased cell growth and this growth was augmented by the expression of mouse PXR and co-treatment with pregnenolone 16α-carbonitrile (PCN), a PXR ligand. In a liver regeneration model using carbon tetrachloride, PCN treatment enhanced the injury-induced increase in the number of Ki-67-positive nuclei as well as Ccna2 and Ccnb1 mRNA levels in wild-type (WT) but not Pxr-null mice. Chronological analysis of this model demonstrated that PCN treatment shifted the maximum cell proliferation to an earlier time point and increased the number of M-phase cells at those time points. In WT but not Pxr-null mice, PCN treatment reduced hepatic mRNA levels of genes involved in the suppression of G0/G1- and G1/S-phase transition, e.g. Rbl2, Cdkn1a and Cdkn1b. Analysis of the Rbl2 promoter revealed that PXR activation inhibited its Forkhead box O3 (FOXO3)-mediated transcription. Finally, the PXR-mediated enhancement of hepatocyte proliferation was inhibited by the expression of dominant active FOXO3 in vitro. The results of the present study suggest that PXR activation stimulates growth factor-mediated hepatocyte proliferation in mice, at least in part, through inhibiting FOXO3 from accelerating cell-cycle progression.

Transactivation of ABCG2 through a novel cis-element in the distal promoter by constitutive androstane receptor but not pregnane X receptor in human hepatocytes.

A previous report demonstrated that treatment of human hepatocytes with phenobarbital, an activator of nuclear receptor constitutive androstane receptor (CAR), increases mRNA levels of an efflux transporter ABCG2, which is involved in the excretion of xenobiotics in liver and intestine. The results suggest that human CAR (hCAR) transactivates human ABCG2 (hABCG2) expression. In this study, we confirmed increase in ABCG2 mRNA levels in human hepatocytes after adenoviral expression of hCAR and treatment with its activator. Reporter assays suggested the existence of an hCAR-responsive element between -8000 and -7485 of hABCG2 promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays identified a DR5 motif (direct repeat separated by five nucleotides) within the region as a binding motif of hCAR/human retinoid X receptor α heterodimer. The introduction of mutations into the DR5 motif resulted in the complete loss of the hCAR-mediated transactivation. Interestingly, human pregnane X receptor, belonging to the same NR1I subfamily as CAR, did not activate any reporter gene containing the DR5 motif. Taken together, our present findings suggest that hCAR transactivates hABCG2 through the DR5 motif located in its distal promoter in human hepatocytes and that the motif prefers hCAR to pregnane X receptor.

Xenobiotic-induced hepatocyte proliferation associated with constitutive active/androstane receptor (CAR) or peroxisome proliferator-activated receptor α (PPARα) is enhanced by pregnane X receptor (PXR) activation in mice.

Xenobiotic-responsive nuclear receptors pregnane X receptor (PXR), constitutive active/androstane receptor (CAR) and peroxisome proliferator-activated receptor α (PPARα) play pivotal roles in the metabolic functions of the liver such as xenobiotics detoxification and energy metabolism. While CAR or PPARα activation induces hepatocyte proliferation and hepatocarcinogenesis in rodent models, it remains unclear whether PXR activation also shows such effects. In the present study, we have investigated the role of PXR in the xenobiotic-induced hepatocyte proliferation with or without CAR activation by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) and phenobarbital, or PPARα activation by Wy-14643 in mice. Treatment with TCPOBOP or phenobarbital increased the percentage of Ki-67-positive nuclei as well as mRNA levels of cell proliferation-related genes in livers as expected. On the other hand, treatment with the PXR activator pregnenolone 16α-carbonitrile (PCN) alone showed no such effects. Surprisingly, PCN co-treatment significantly augmented the hepatocyte proliferation induced by CAR activation with TCPOBOP or phenobarbital in wild-type mice but not in PXR-deficient mice. Intriguingly, PXR activation also augmented the hepatocyte proliferation induced by Wy-14643 treatment. Moreover, PCN treatment increased the RNA content of hepatocytes, suggesting the induction of G0/G1 transition, and reduced mRNA levels of Cdkn1b and Rbl2, encoding suppressors of cell cycle initiation. Our present findings indicate that xenobiotic-induced hepatocyte proliferation mediated by CAR or PPARα is enhanced by PXR co-activation despite that PXR activation alone does not cause the cell proliferation in mouse livers. Thus PXR may play a novel and unique role in the hepatocyte/liver hyperplasia upon exposure to xenobiotics.

Constitutive androstane receptor transactivates the hepatic expression of mouse Dhcr24 and human DHCR24 encoding a cholesterogenic enzyme 24-dehydrocholesterol reductase.

Phenobarbital treatment has long been known to influence serum and hepatic cholesterol levels in rodents and humans. Constitutive androstane receptor (CAR), a member of the nuclear receptor superfamily, mediates various biological actions of phenobarbital. We have thus investigated whether CAR transactivates cholesterogenic genes in livers. Activation of CAR in mouse livers and cultured human hepatocytes increased mRNA levels of mouse Dhcr24 and human DHCR24, both of which encode 24-dehydrocholesterol reductase (DHCR24) catalyzing the last step of cholesterol biosynthesis. CAR transactivated the expression of these genes in reporter assays with cultured hepatoma cells. Furthermore, we have identified a DR4 (direct repeat separated by 4 nucleotides) motif in the human DHCR24 distal promoter as a binding site of CAR/retinoid X receptor α (RXRα) heterodimer. We have also demonstrated that the heterodimer of pregnane X receptor (PXR)/ RXRα binds to the DR4 motif and that human DHCR24 reporter gene is transactivated by the ligand-activated PXR. These results suggest a role of xenobiotic-responsive nuclear receptor CAR, and also possibly PXR, in cholesterol biosynthesis in the liver of mice and humans.