Discussion and Conclusions

CAR plays an important role in the control of both endogenous and xenobiotic compound metabolism. It was reported that CAR activation by TCPOBOP leads to hepatomegaly and an increase in both hepatocyte growth and proliferation in mice. Since YAP is a potent regulator of liver size and tissue homeostasis, its role in the hepatoproliferative effects of CAR was investigated. Most recently, YAP/TEAD activation was found to play a role in CAR-dependent proliferation of murine hepatocytes (Abe et al., 2018). However, the role of YAP in CAR-induced hepatomegaly and liver regeneration, and the relationship between CAR and YAP remains unknown. In the current study, CAR activation significantly promoted nuclear translocation of YAP and directly interacted with YAP, which consequently promotes hepatomegaly and liver regeneration thus indicating a potential role for YAP in CAR-induced hepatomegaly and liver regeneration. These findings provide new insights for understanding the physiological functions of CAR.
It was reported that CAR activation via TCPOBOP can induce robust hepatocyte proliferation and hepatomegaly in mouse livers and promote liver regeneration after liver tissue loss (Costa, Kalinchenko, Tan & Wang, 2005; Tschuor et al., 2016). In the current study, CAR-induced hepatomegaly and liver regeneration was confirmed in WT and the PHx mouse model. All mice were male in order to eliminate the gender difference, since CAR can be activated by estrogens, although to a lesser degree than by exogenous ligands such as TCPOBOP (Kachaylo, Pustylnyak, Lyakhovich & Gulyaeva, 2011; Min, Kim, Bae, Petz & Kemper, 2002). CAR-induced hepatomegaly and liver regeneration in this study is mainly caused by hepatocyte enlargement and proliferation. Hepatocytes proliferation can be observed only in the PV area, while the increase of cell size was only shown in the CV area. A possible reason for this phenomenon is that CAR activation induces the expression of enzymes and perhaps the enlargement of subcellular structures, since enzymes related to glycogen synthesis, glycolysis, lipogenesis, ketogenesis, and detoxication are preferentially situated in the CV area (Jiang et al., 2019; Jungermann & Katz, 1989).
YAP is a key downstream factor of the Hippo signaling pathway, which is a crucial regulator of organ size and tissue homeostasis (Kowalik et al., 2011). A previous study demonstrated that another nuclear receptor, pregnane X receptor (PXR), can also induce liver enlargement and promote liver regeneration via activation of YAP (Jiang et al., 2019). Both PXR and CAR belong to the superfamily of nuclear receptors (Chai, Zeng & Xie, 2013), suggesting that CAR may activate or interact YAP as well. A previous cell-based study suggested that there is a functional crosstalk between CAR and YAP in the nucleus of hepatocytes, but there is no evidence suggesting a functional relationship between CAR and YAP (Abe et al., 2018). In this study, CAR activation can significantly promote the nuclear translocation of YAP and then upregulate the downstream targets of YAP. Furthermore, Co-IP assays showed a potential protein-protein interaction between CAR and YAP, which suggest the important role of YAP in CAR-induced liver enlargement and regeneration. However, additional evidence such as surface plasmon resonance and isothermal titration calorimetry is needed to confirm their interactions, and to determine the amino acids that lead to the CAR and YAP interaction.
Yap liver-specific knockout mice revealed that CAR-induced hepatomegaly was only partially dependent on the YAP pathway, suggesting other regulatory factors may be involved in CAR-induced liver enlargement. Many regulators were reported to be involved in CAR activation-induced hepatomegaly such as C-Myc, FOXM1, CTNNB1, EGFR and MET. According to previous reports, the activation of CAR can still promote hepatomegaly in Ctnnb1 knockout mice (Ganzenberg, Singh & Braeuning, 2013). The depletion of C-Myc also only partially restored the enlargement of liver (Blanco-Bose et al., 2008). Similarly, liver enlargement can still be observed in EGFRi+MET double knockout mice after treatment with TCPOBOP for 10 days (Bhushan et al., 2019). These studies indicated complicated mechanisms in CAR-induced liver enlargement and regeneration process. In the current study, the mRNA and protein levels of related factors involved in hepatocytes enlargement and proliferation were measured. In WT mice, the expression of C-Myc and MET were significantly upregulated after treated by TCPOBOP. Interestingly, the level of these proteins were even higher in TCPOBOP-treated Yap -/-mice than WT mice. We hypothesize that these proteins were upregulated to a higher extent to compensate the proliferative response reduced by the loss of Yap . CTNNB1 was not significantly changed consistent with a previous report (Ganzenberg, Singh & Braeuning, 2013), but can be downregulated inYap-/- mice, and then be induced by the activation of CAR, suggesting that there may be a crosstalk between the CTNNB1 and YAP pathways. On the other hand, a previous study revealed that PXR can also promote hepatomegaly and liver regeneration via YAP pathway. However, the activation of PXR failed to induce liver enlargement and liver cell fate change when YAP was depleted, indicating that PXR-induced hepatomegaly is totally in a YAP dependent manner (Jiang et al., 2019).
YAP downstream targets such as CTGF, ANKRD1 were upregulated even inYap-/- mice. We hypothesize that these proteins might be regulated by other factors simultaneously. CTGF is a member of the CCN family, which is involved in diverse biological processes such as cell adhesion, proliferation, and angiogenesis. Previous studies reported that CTGF can be regulated by the Wnt/β-catenin signaling pathway (Deng et al., 2007; Li et al., 2012). Meanwhile, it is also a potential target of Wnt and BMP signaling (Luo et al., 2004). ANKRD1 is a potent regulator of early cardiac development, it can be significantly upregulated in cardiac hypertrophy and heart failure (Kojic et al., 2010). ANKRD1 was cooperatively induced by both the TGF-β and Wnt pathways in normal murine mammary gland epithelial cells. The upregulation of ANKRD1 can be observed in models of Wnt/β-catenin-induced tumors (Labbe et al., 2007). Our results suggested that the β-catenin signaling pathway was activated inYap-/- mice, and thus CTGF and ANKRD1 were still significantly upregulated in Yap-/- mice.
Cyclin D and cyclin E are two major classes of cyclins expressed in mammalian cells during the G1 phase of the cell cycle (Geng et al., 1999). Protein levels of CCND1 and CCNE1 were upregulated significantly at both time points in TCPOBOP-treated WT mice. This suggested that CAR activation induces hepatocyte proliferation by promoting G1-S transition. Since DNA synthesis in hepatocytes was terminated 4-5 days after PHx, the liver mass was restored so the proliferative response was much weaker at Day 5 (Jiang et al., 2019). Thus, in the PHx vehicle group, the expression of CCNE1 was downregulated at Day 5 while CCND1 didn’t change significantly.
Liver possesses the ability to regenerate in response to injury. Liver regeneration can be defined as compensatory hyperplasia with the remaining liver tissue expands to meet the metabolic needs of the organism, however, the expanding liver does not regain its original gross anatomical structure. The pathways involved in liver regeneration include cytokines, growth factors, and metabolic networks. The most completely studied model is that of liver regeneration following partial hepatectomy, which 3 of 5 liver lobes (2/3 of the liver mass) is removed (Mao, Glorioso & Nyberg, 2014). It was reported that CAR deficiency impaired liver regeneration, while the CAR activation through pharmacological means is sufficient to prevent or rescue the experimental SFSS (small-for-size-syndrome), which is a modified version of extended hepatectomy in mice (eHx, 86% removed) (Tschuor et al., 2016). The present study showed that YAP, a critical factor of liver regeneration, was activated in TCPOBOP-promoted liver regeneration in PHx model, which aids the liver to regain its normal functions.
In summary, the current study demonstrate that CAR activation can promote liver enlargement and liver regeneration partially by inducing nuclear translocation of YAP and interaction with the YAP signalling pathway. These findings provide new insights for understanding the physiological functions of CAR and suggests the potential for manipulation of liver size. CAR can be a potential target to promote liver regeneration after liver surgery and rescue SFSS during the transplantation of liver. The investigation of protein-protein interaction between CAR and YAP may provide therapeutic strategy that can avoid the side effect of CAR activation and promote its clinical use in the future.
Acknowledgements: The work was supported by the Natural Science Foundation of China (Grants: 81973392), the National Key Research and Development Program (Grant: 2017YFE0109900), the Natural Science Foundation of Guangdong (Grant: 2017A030311018), the 111 project (Grant: B16047), the Key Laboratory Foundation of Guangdong Province (Grant: 2017B030314030), the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01Y093), and the National Engineering and Technology Research Center for New drug Druggability Evaluation (Seed Program of Guangdong Province, 2017B090903004).
Conflict of interest: There are no competing interests.
Author contribution: H.B. and M.H. conceived the project. Y.G., S.F., X.Y., R.W., and J.T. performed the experiments. H.L. and S.Z. contributed to the animal models. Y.J., X.Y., and F.J.G participated in the scientific discussion and research design.