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.