ABSTRACT
Constitutive androstane receptor (CAR), a nuclear receptor predominantly expressed in the liver, is activated by diverse chemicals and induces hepatocyte proliferation and hepatocarcinogenesis in rodents. However, the underlying mechanism responsible for CAR-dependent hepatocyte proliferation remains unclear. Importantly, this phenomenon has not been observed in the human liver. This study aimed to investigate the molecular mechanism underlying CAR-induced hepatocyte proliferation and to explore the species differences in hepatocyte proliferation between humans and rodents. Treatment of mice with the CAR activator TCPOBOP induced hepatocyte proliferation and nuclear accumulation of yes-associated protein (YAP), a known liver cancer inducer. This induction was abolished in CAR-knockout mice. Exogenously expressed YAP in cultured cells was accumulated in the nucleus by the coexpression with mouse CAR but not human CAR. Pull-down analysis of recombinant proteins revealed that mouse CAR interacted with YAP, whereas human CAR did not. Further investigations using YAP deletion mutants identified the WW domain of YAP as essential for interacting with CAR and showed that the PY motif (PPAY) in mouse CAR was crucial for binding to the WW domain, whereas human CAR with its mutated motif (PPAH) failed to interact with YAP. A mouse model harboring the Y150H mutation (PPAY to PPAH) in CAR displayed drastically attenuated TCPOBOP-induced hepatocyte proliferation and nuclear accumulation of YAP. CAR induces the nuclear accumulation of YAP through the PY motif-WW domain interaction to promote hepatocyte proliferation. The absence of this interaction in human CAR contributes to the lack of CAR-dependent hepatocyte proliferation in human livers.
Subject(s)
Constitutive Androstane Receptor , Rodentia , Animals , Humans , Mice , Cell Proliferation , Hepatocytes/metabolism , Liver/metabolism , Species SpecificityABSTRACT
Pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are nuclear receptors that are highly expressed in the liver and activated by numerous chemicals. While CAR activation by its activators, such as phenobarbital (PB), induces hepatocyte proliferation and liver carcinogenesis in rodents, it remains unclear whether PXR activation drives liver cancer. To investigate the influence of PXR activation on liver carcinogenesis, we treated mice with the PXR activator pregnenolone 16α-carbonitrile (PCN) with or without PB following tumor initiation with diethylnitrosamine (DEN). After 20 weeks of treatment, preneoplastic lesions detected by immunostaining with an anti-KRT8/18 antibody were observed in PB-treated but not PCN-treated mice, and PCN cotreatment augmented the formation of preneoplastic lesions by PB. After 35 weeks of treatment, macroscopic observations indicated that PB-treated and PB/PCN-cotreated mice had increased numbers of liver tumors compared to control and PCN-treated mice. In the pathological analyses of liver sections, all the mice in the PB and PB/PCN groups developed carcinoma and/or eosinophilic adenoma, but in the PB/PCN group, the multiplicity of carcinoma and eosinophilic adenoma was significantly reduced and the size of carcinoma showed a tendency to decrease. No mouse in the control or PCN-treated group developed such tumors. Differentially expressed gene (DEG) and gene set enrichment analyses in combination with RNA sequencing suggested the increased expression of genes related to epithelial-mesenchymal transition (EMT) in mice cotreated with PCN and PB compared to those treated with PB alone. Changes in the hepatic mRNA levels of epithelial marker genes supported the results of the transcriptome analyses. In conclusion, the present results suggest that PXR activation does not promote hepatocarcinogenesis in contrast to CAR and rather attenuates CAR-mediated liver cancer development by suppressing the EMT of liver cancer cells in rodents.
Subject(s)
Liver Neoplasms/chemically induced , Phenobarbital/pharmacology , Pregnane X Receptor/drug effects , Pregnenolone Carbonitrile/pharmacology , Animals , Carcinogenesis/drug effects , Cell Proliferation/drug effects , Constitutive Androstane Receptor , Hepatocytes/drug effects , Liver/drug effects , Liver/pathology , Liver Neoplasms/pathology , Male , Mice , Mice, Inbred C3H , Pregnane X Receptor/metabolism , Receptors, Cytoplasmic and Nuclear/drug effects , Receptors, Cytoplasmic and Nuclear/metabolism , Sequence Analysis, RNA , Time FactorsABSTRACT
Despite evidence that tripeptide glycyl-Ê-histidyl-Ê-lysine (GHK) is an endogenous antioxidant, its mechanism and importance are not fully understood. In the present study, the ability of GHK to reduce levels of reactive oxygen species (ROS) in Caco-2 cells was evaluated by flow cytometry with the oxidation-sensitive fluorescent dye 2',7'-dichlorodihydrofluorescein diacetate. Further, types of ROS diminished by GHK were assessed by utilizing an electron spin resonance (ESR) spin-trapping technique. GHK reduced the tert-butyl hydroperoxide-induced increase in ROS levels in Caco-2 cells at concentrations of 10 µM or less. Experiments utilizing an ESR spin-trapping technique revealed that, among hydroxyl (·OH), superoxide (O2-·), and peroxyl (ROO·) radicals generated by respective chemical reaction systems, GHK diminished signals of both ·OH and ROO·, but not O2-·. Additionally, the GHK effect on the signal of ·OH was much stronger than those of other well-known antioxidative, endogenous peptides, carnosine and reduced glutathione. These results suggest that GHK can function as an endogenous antioxidant in living organisms, possibly by diminishing ·OH and ROO·.
