Elsevier

Toxicology

Volume 321, 3 July 2014, Pages 73-79
Toxicology

CAR-mediated repression of Foxo1 transcriptional activity regulates the cell cycle inhibitor p21 in mouse livers

https://doi.org/10.1016/j.tox.2014.04.003Get rights and content

Highlights

  • CAR activation decreased the level of Foxo1 in mouse livers.

  • CAR activation decreased the level of p21 in mouse livers.

  • CAR activation inhibited Foxo1 transcriptional activity in mouse livers.

Abstract

1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), an agonist of constitutive androstane receptor (CAR), is a well-known strong primary chemical mitogen for the mouse liver. Despite extensive investigation of the role of CAR in the regulation of cell proliferation, our knowledge of the intricate mediating mechanism is incomplete. In this study, we demonstrated that long-term CAR activation by TCPOBOP increased liver-to-body weight ratio and decreased tumour suppressor Foxo1 expression and transcriptional activity, which were correlated with reduced expression of genes regulated by Foxo1, including the cell-cycle inhibitor Cdkn1a(p21), and upregulation of the cell-cycle regulator Cyclin D1. Moreover, we demonstrated the negative regulatory effect of TCPOBOP-activated CAR on the association of Foxo1 with the target Foxo1 itself and Cdkn1a(p21) promoters. Thus, we identified CAR-mediated repression of cell cycle inhibitor p21, as mediated by repression of FOXO1 expression and transcriptional activity. CAR-FOXO1 cross-talk may provide new opportunities for understanding liver diseases and developing more effective therapeutic approaches to better drug treatments.

Introduction

The constitutive androstane receptor (CAR), which is expressed primarily in the liver, was initially characterised as a xenosensor that regulates responses to xenochemicals. CAR mediates the up-regulation of xenobiotic/drug-metabolising enzymes, increasing the metabolic capability of the liver to protect cells from xenochemical toxicity (Kachaylo et al., 2011). Moreover, CAR regulates other physiologically important enzymes in the liver. For instance, CAR has been demonstrated not only to be a xenosensor but also to play a role in endogenous energy metabolism. Phosphoenolpyruvate carboxykinase (Pck1) and glucose-6-phosphatase (G6pc), key gluconeogenic genes are repressed in response to CAR activators, and this repression is CAR dependent (Ueda et al., 2002, Kachaylo et al., 2012, Yarushkin et al., 2013). CAR activation by xenobiotics also causes liver hyperplasia and hepatomegaly in the short term (Huang et al., 2005, Blanco-Bose et al., 2008). Long-term treatments with these compounds cause liver tumours in rodents. Studies using CAR-null mice have demonstrated that CAR activation is an essential requirement for liver tumour development via a nongenotoxic mode of action, apparently through the induction of cell proliferation and suppression of apoptosis (Yamamoto et al., 2004, Huang et al., 2005). Indeed, CAR activation is associated with the increased expression of a number of cell cycle regulators, including Cyclin D1, Mdm2, cMyc, Gadd45β, Cdkn1a and others (Ledda-Columbano et al., 2003, Columbano et al., 2005, Yamamoto and Negishi, 2008, Yamamoto et al., 2010, Huang et al., 2005, Blanco-Bose et al., 2008, Kazantseva et al., 2013). However, the entire mechanism of the liver tumour formation promoted by CAR in rodents has not been fully elucidated.

Forkhead box O1 (Foxo1) is a member of the superfamily of transcription factors that share a highly conserved DNA-binding FOX domain (Zhang et al., 2011). Foxo1 has been demonstrated to bind to various forms of consensus sequences (Guo et al., 1999, O’Brien et al., 2001, Nakae et al., 2008, Armoni et al., 2006). Specifically, Foxo1 promotes glucose production in the liver through the transcriptional regulation of target genes such as G6pc and Pck1 (Schmoll et al., 2000, Yeagley et al., 2001). FOXO1 is emerging as a master signalling regulator that controls many physiological and pathological processes. There are many parallels in the functions of Foxo1 and p53, a well-known tumour suppressor. Both proteins regulate cellular differentiation, growth, survival, cell cycle, metabolism and stress (Zhang et al., 2011). Cell cycle arrest by Foxo1 and p53 occurs through inducing their target genes such as Cdkn1a(p21) and Cdkn1b(p27). Moreover, Foxo1, such as p53, has been demonstrated to be involved in Bcl-2 family gene expression regulation. Thus, an inactivation of Foxo1 appears to be a crucial step in tumourigenesis.

Foxo1 is regulated at multiple levels, which include phosphorylation, ubiquitylation, acetylation. Foxo1 is a downstream target of the PI3K/Akt signalling pathway, which is an essential pathway for cell survival and growth during development and tumourigenesis. Upon activation, Akt phosphorylates Foxo1, leading to its nuclear exclusion and increased proteosomal degradation that dampens its transcriptional regulation of target genes (Jackson et al., 2000, Matsuzaki et al., 2003). Moreover, interaction with other proteins also regulates the transcriptional activity of FOXO1. For example, the PPAR-γ coactivator 1 (Pgc1) interacts with Foxo1 and stimulates gluconeogenesis in the liver (Puigserver et al., 2003). On the other hand, activated CAR binds to Foxo1 and prevents its binding to the gluconeogenic genes promoters, which results in transcriptional repression of the target genes G6pc and Pck1 (Kodama et al., 2004). As cell proliferation is regulated by both CAR and Foxo1, we examined if the liver hyperplasia promoted by CAR activation occurs through Foxo1 repression. This target was studied using a well-known strong primary chemical mitogen for the liver, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). TCPOBOP, which is an agonist of mouse CAR, is a nongenotoxic hepatocarcinogen by itself and is a potent tumour promoter when combined with genotoxic agents. This mitogen produces rapid direct liver hyperplasia and hepatomegaly in the absence of injury (Ledda-Columbano et al., 2000, Huang et al., 2005, Blanco-Bose et al., 2008, Tian et al., 2011).

Section snippets

Chemicals

ТСРОВОР was obtained from Sigma–Aldrich (MO, USA). 3α-Hydroxy-5α-androstanol (Andr) was obtained from Steraloids (USA). All other analytical grade chemicals and solvents were obtained from commercial sources.

Experimental animals

Male C57BL mice (25–30 g) were supplied by the Institute of Clinical Immunology SB RAMS (Novosibirsk, Russia). Animals were acclimated for 1 week and allowed free access to food and water. All experimental procedures were approved by the Animal Care Committee for the Institute of Molecular

Effects of TCPOBOP on CAR target Cyp2b10

The Cyp2b10 gene is a commonly used biomarker to measure CAR activation. Total RNA was isolated from mouse livers 8 weeks after treatment with TCPOBOP or the vehicle, and the mRNA level of Cyp2b10 was measured by real-time PCR. As expected from previous results, hepatic Cyp2b10 gene expression, which is a typical CAR target gene, was induced by TCPOBOP (Fig. 1A). Next, we examined the effect of long-term TCPOBOP treatment on the protein level in the liver (Fig. 1B). Hepatic Cyp2b10 was

Discussion

The ability of nuclear receptors to transduce extracellular signal into fast changes in gene expression proposes these transcription factors key players coordinate in different cell processes, including cell proliferation. CAR effects after its translocation to the nucleus appear to contribute to molecular pathways underlying hepatocyte proliferation. CAR can modulate pathways involved in liver regeneration after partial hepatectomy, induce direct hyperplasia and has profound effects on hepatic

Conclusion

In conclusion, our findings demonstrated a novel role of CAR-Foxo1 cross-talk in the regulation of cell proliferation through CAR-mediated repression of the tumour suppressor Foxo1 and the cell cycle inhibitor Cdkn1a(p21). A detailed understanding of CAR-Foxo1 cross-talk will provide new opportunities for developing more effective therapeutic approaches to treat liver diseases.

Conflict of interest

The authors declare that there are no conflicts of interest.

Transparency document

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Acknowledgments

This study was partially supported by RFBR, research project 14-04-01350 a.

References (46)

  • S. Pelengaris et al.

    The many faces of c-MYC

    Arch. Biochem. Biophys.

    (2003)
  • V. Pustylnyak et al.

    Effect of several analogs of 2,4,6-triphenyldioxane-1,3 on CYP2B induction in mouse liver

    Chem. Biol. Interact.

    (2011)
  • S. Ramaswamy et al.

    A novel mechanism of gene regulation and tumor suppression by the transcription factor FKHR

    Cancer Cell.

    (2002)
  • D. Schmoll et al.

    Regulation of glucose-6-phosphatase gene expression by protein kinase Balpha and the forkhead transcription factor FKHR. Evidence for insulin response unit-dependent and -independent effects of insulin on promoter activity

    J. Biol. Chem.

    (2000)
  • J. Seoane et al.

    Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation

    Cell

    (2004)
  • D. Yeagley et al.

    Gene- and activation-specific mechanisms for insulin inhibition of basal and glucocorticoid-induced insulin-like growth factor binding protein-1 and phosphoenolpyruvate carboxykinase transcription. Roles of forkhead and insulin response sequences

    J. Biol. Chem.

    (2001)
  • X. Zhang et al.

    Akt, FoxO and regulation of apoptosis

    Biochim. Biophys. Acta

    (2011)
  • J.H. Albrecht et al.

    Involvement of p21 and p27 in the regulation of CDK activity and cell cycle progression in the regenerating liver

    Oncogene

    (1998)
  • B. Al-Mubarak et al.

    Synaptic NMDAR activity suppresses FOXO1 expression via a cis-acting FOXO binding site: FOXO1 is a FOXO target gene

    Channels (Austin)

    (2009)
  • W.E. Blanco-Bose et al.

    C-Myc and its target FoxM1 are critical downstream effectors of constitutive androstane receptor (CAR) mediated direct liver hyperplasia

    Hepatology

    (2008)
  • A. Columbano et al.

    Gadd45beta is induced through a CAR-dependent, TNF-independent pathway in murine liver hyperplasia

    Hepatology

    (2005)
  • H. Daitoku et al.

    Regulation of PGC-1 promoter activity by protein kinase B and the forkhead transcription factor FKHR

    Diabetes

    (2003)
  • L.E. Greenbaum

    Cell cycle regulation and hepatocarcinogenesis

    Cancer Biol. Ther.

    (2004)
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