Introduction

The constitutive androstane receptor (CAR, NR3I1) is a xenobiotic-sensing nuclear receptor and a member of the nuclear receptor superfamily (Konno, Negishi & Kodama, 2008). Like other nuclear receptors, CAR is composed of a DNA-binding domain, a large carboxy-terminal ligand-binding domain (LBD), and a poorly conserved amino-terminal domain (Willson & Kliewer, 2002). CAR is localized in the cytoplasm by forming a complex with HSP90 and the cytoplasmic CAR retention protein (CCRP). Once activated, CAR dissociates from the cytoplasmic complex and is translocated into the nucleus where it forms a heterodimer with retinoid X receptor alpha (RXRα, ΝΡ2Β1) and activates transcription of its target genes (Kodama & Negishi, 2006).
CAR activates transcription of its target genes encoding various enzymes and proteins involved in drug metabolism and transport when exposure to xenobiotics (Konno, Negishi & Kodama, 2008; Wei, Zhang, Egan-Hafley, Liang & Moore, 2000). In addition to xenobiotics, CAR can also regulate the elimination of toxic endobiotics such as bilirubin and bile acids, so one major function of CAR is detoxification (Eloranta & Kullak-Ublick, 2005; Sugatani et al., 2002). However, recent studies have also revealed a key role for CAR in various physiological and pathophysiological processes occurring in the liver including gluconeogenesis, metabolism of fatty acids, metabolism of lipids, hormonal regulation, proliferation of hepatocytes, and hepatocarcinogenesis (Kachaylo, Pustylnyak, Lyakhovich & Gulyaeva, 2011). CAR is also involved in metabolic diseases including jaundice, cholestasis, thyroid homeostasis and obesity (Dash et al., 2014; Qatanani & Moore, 2005).
TCPOBOP (1,4-Bis [2-(3,5-dichloropyridyloxy)] benzene), a strong agonist of CAR, was reported to induce robust hepatocyte proliferation and hepatomegaly in mice livers (Costa, Kalinchenko, Tan & Wang, 2005). Moreover, the rapid proliferation response induced by TCPOBOP can protect liver from failure even after massive tissue loss by 91% hepatectomy (Tschuor et al., 2016). The mechanism of TCPOBOP-induced hepatomegaly was studied in previous reports. C-Myc and FOXM1 mediated proliferative programs are key mediators of TCPOBOP-CAR induced direct liver hyperplasia (Blanco-Bose et al., 2008). β-catenin (CTNNB1) deficiency decreases the impact of CAR activation on hepatocytes proliferation (Ganzenberg, Singh & Braeuning, 2013). Disruption of EGFR and MET signaling leads to a dramatic impairment of the TCPOBOP-induced proliferative response without altering CAR activation (Bhushan et al., 2019).
Yes-associated protein (YAP) is a key downstream factor of Hippo signaling pathway, which is a potent regulator of organ size and tissue homeostasis (Kowalik et al., 2011). When Hippo signaling is ON, YAP is phosphorylated by LATS1/2 and binding with 14-3-3 in cytoplasm. When Hippo signaling is OFF, YAP can be dephosphorylated and translocated into nucleus where it binds with TEAD to activate transcription of genes involved in cell survival, growth, and proliferation (Patel, Camargo & Yimlamai, 2017). According to previous studies, YAP increases organ size and causes aberrant tissue expansion in mice. YAP activation reversibly increases liver size more than 4-fold (Camargo et al., 2007). Removal ofMst1 and Mst2 (the upstream of YAP) in liver, which activates YAP, results in significant liver enlargement and dysplasia (Song et al., 2010). In addition, YAP also participates in liver regeneration after partial hepatectomy (PHx). Hepatocyte nuclear accumulation of YAP increased in parallel with hepatocyte proliferative activity after PHx. Deleting Yap from hepatocytes blocked the nuclear accumulation of pSmad2 and EMT-like response, as well as their proliferation (Oh, Swiderska-Syn, Jewell, Premont & Diehl, 2018). Inhibition of MST1 and MST2 augments liver repair and regeneration by activating the downstream effector YAP and promoting cell growth (Fan et al., 2016).
Most recently, YAP/TEAD activation was found to participate in CAR-dependent proliferation of murine hepatocytes (Abe et al., 2018). However, this study used an in vitro cell-based system and an animal experiment of verteporfin (an inhibitor of YAP/TEAD interaction) treatment. The mechanistic influence of CAR activation on YAP signaling has not been clarified and the relationship between CAR and YAP remains unknown. Therefore, the current study aims to elucidate the role of YAP in CAR-induced hepatomegaly using liver-specific YAP-deficient mice, to investigate whether YAP is associated with CAR-promoted liver regeneration, and further explore the relationship between CAR and YAP.