Role of YAP Activation in Nuclear Receptor CAR-Mediated Proliferation of Mouse Hepatocytes

Abstract

Constitutive androstane receptor (CAR) is a xenobiotic-responsive nuclear receptor that is highly expressed in the liver. CAR activation induces hepatocyte proliferation and hepatocarcinogenesis in rodents, but the mechanisms remain unclear. In this study, we investigated the association of CAR-dependent cell proliferation with Yes-associated protein (YAP), which is a transcriptional cofactor controlling organ size and cell growth through the interaction with various transcriptional factors including TEA domain family member (TEAD). In mouse livers, 1,4-bis-(2-[3,5-dichloropyridyloxy])benzene (TCPOBOP) (a mouse CAR [mCAR] activator) treatment increased the nuclear YAP accumulation and mRNA levels of YAP target genes as well as cell-cycle related genes along with liver hypertrophy and verteporfin (an inhibitor of YAP/TEAD interaction) cotreatment tended to attenuate them. Furthermore, in cell-based reporter gene assays, CAR activation enhanced the YAP/TEAD-dependent transcription. To investigate the role of YAP/TEAD activation in the CAR-dependent hepatocyte proliferation, we sought to establish an in vitro system completely reproducing CAR-dependent cell proliferation. Since CAR was only slightly expressed in cultured mouse primary hepatocytes compared with mouse livers and no proliferation was observed after treatment with TCPOBOP, we overexpressed CAR using mCAR expressing adenovirus (Ad-mCAR-V5) in mouse primary hepatocytes. Ad-mCAR-V5 infection and TCPOBOP treatment induced hepatocyte proliferation. Similar results were obtained with immortalized normal mouse hepatocytes as well. In the established in vitro system, CAR-dependent proliferation was strongly inhibited by Yap knockdown and completely abolished by verteporfin treatment. Our present results obtained in in vivo and in vitro experiments suggest that YAP/TEAD activation plays key roles in CAR-dependent proliferation of murine hepatocytes.

Role of Nuclear Receptors PXR and CAR in Xenobiotic-Induced Hepatocyte Proliferation and Chemical Carcinogenesis

“…39) This hypothesis seemed too easy and we soon found that this was not the case but the results were very surprising and interesting (see below), which drove us to start a series of experiments on the involvement of PXR in hepatocyte proliferation. 31,32,34) Single treatment of mice with TCPOBOP increased the number of proliferation marker Ki-67-positive hepatocytes 48 h after the treatment whereas PCN did not, as expected. These findings were confirmed by determining mRNA levels of cell cycle-related genes such as Ccna2, Ccnb1, Mcm2, or Mki67.…”

“…To investigate this possibility, we carried out a series of experiments using in vitro and in vivo models. 31,32) We first used immortalized mouse normal hepatocytes, AML12 cells, as an in vitro model. 32) When AML12 cells are cultured in the absence of serum for 48 h, they become synchronized at the G0 phase, and we are able to make the cells enter the cell cycle by culturing them in medium containing serum.…”

“…As expected, PCN co-treatment enhanced CCl 4 -induced increase in the number of Ki-67-positive hepatocytes as well as hepatic mRNA levels of cell cycle-related genes, and these enhancements were not observed in Pxr-null mice, suggesting that the enhancement is PXR-dependent. 32) To confirm these in vivo enhancing effects of PXR, we investigated the influence of PXR activation on hepatocyte proliferation induced by fibroblast growth factor 19 (FGF19), which is reported to induce hepatocyte proliferation in rodents. 53,54) Mice were treated subcutaneously with recombinant FGF19 with or without PCN co-treatment for 3 d and hepatocyte proliferation was investigated.…”

“…In this manuscript, I will review the background and our recent results on the role of PXR in hepatocyte proliferation and the mechanism for CARmediated hepatocyte proliferation. [31][32][33][34]…”

Is This the Time to Reconsider the Names for Xenobiotic Nuclear Receptors?

“…Kowalik et al showed that activation of CAR increased nuclear translocation of YAP, which, for the first time, suggests the interaction of the xenobiotic nuclear receptor family member with the Hippo–YAP pathway . Furthermore, Abe et al showed that knockdown or pharmacological inhibition of YAP drastically diminished the CAR‐dependent cell growth and induction of the cell proliferation marker genes . Interestingly, similar to PXR, CAR activation–induced cell proliferation and drug disposition are also detached, as shown by a recent publication from Bhushan et al However, a direct physical interaction between CAR and YAP has not been determined from these studies.…”