The CAR nuclear receptor and hepatocyte proliferation
Authors: Robert H. Costa, Vladimir V. Kalinchenko, Yongjun Tan, I-Ching Wang
Journal: Hepatology (2005)

Liver regeneration induced by two-thirds partial hepatectomy (PHx) results in synchronous induction of hepatocyte DNA replication (S-phase) and mitosis (M-phase). This proliferative response is initiated by release of the cytokines tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) and the growth factors tumor growth factor α (TGFα) and hepatocyte growth factor (HGF), all of which mediate reentry of terminally differentiated hepatocytes into the cell cycle.1-4 This hepatocyte proliferative response during liver regeneration requires activation of the Ras-mitogen activated protein kinase (MAPK) signaling pathway and is coincident with rapid transcriptional induction of immediate early transcription factors including the basic leucine zipper (bZIP) c-Jun and c-Fos heterodimers (AP1) and the basic-helix-loop-helix (bHLH) c-Myc protein. Furthermore, the latent immediate early transcription factors nuclear factor κB (NFκB), CCAAT/Enhancer Binding Protein β (C/EBPβ) proteins and signal transducers and activators of transcription 3 (Stat3) are activated through phosphorylation dependent signaling pathways.2, 5-7 Several lines of evidence establish a critical role for TNFα and its rapidly activated downstream target, IL-6, in the transition of hepatocytes into the cell cycle following PHx. Activation of AP1 and NFκB transcription factors, as well as DNA synthesis, can be inhibited by pre-treatment of the mice with antibodies against TNFα,8 suggesting that this cytokine plays a central role in activating transcription factors required for initiation of liver regeneration. Following PHx, IL-6-deficient−/− mice or TNF receptor type 1 (TNFR1)−/− mice exhibited a 70% reduction in hepatocyte DNA replication and the proliferation defect in both knockout mice are corrected after an intraperitoneal (IP) injection of IL-6 to these mice prior to surgery.9, 10 These results indicate that regulation of IL-6 by the TNFα–NFκB signaling pathway is of critical importance in initiating the liver regeneration process in response to PHx. This decrease in proliferation of regenerating hepatocytes was accompanied by diminished induction of immediate early transcription factors including Stat3, c-Jun, c-Fos, NFκB and c-Myc and a failure to activate expression of Cyclin D and Cyclin E, which are required for hepatocyte entry into S-phase (see Fig. 1). In this issue of HEPATOLOGY, the article by Columbano et al.11 builds on several observations regarding the molecular basis of hepatocyte proliferation in response to primary mitogens. The authors show that hepatocyte proliferation in TNFR1−/−TNFR2−/− double knockout mice, which are normally nonresponsive to surgically induced liver regeneration,10, 12 can be stimulated by the synthetic ligand of the constitutive androstane receptor (CAR), 1,4-bis- (2-(3, 5, -dichloropyridyloxy)) benzene (TCPOBOP), and that this proliferation occurred without activation of NFκB and Stat3 transcription factors. These results suggest that CAR agonists are potentially useful to stimulate hepatocyte proliferation during liver transplantation of suboptimal cadaver donor livers that would normally not be suitable for liver transplantation. Regulation of cell cycle genes in proliferating hepatocytes following partial hepatectomy induced liver regeneration. Blue arrows represent cell cycle signaling, green arrows represent positive transcriptional regulation, red lines represent protein inhibition or negative regulation, orange arrows represent phosphorylation, and light orange arrows represent dephosphorylation. Circle represents the different stages of the cell cycle including Gap1 (G1), DNA replication (S-phase), Gap2 (G2), and mitosis (M-phase). The cytokine (TNFα and IL-6) and growth factor (TGFα and HGF) induction of the Ras-MAPK signaling pathway stimulate expression of immediate early transcription factors (AP1, C/EBPβ, NFκB, and Stat3). These transcription factors regulate the temporal expression of the Cyclin D1 regulatory subunit, which activates its corresponding Cyclin-dependent kinase (Cdk) 4 and 6 through complex formation.13-15 TNFα–NFκB signaling pathway also stimulates expression of Gadd45β gene, which is involved in preventing apoptosis. Progression into DNA replication (S-phase) requires phosphorylation of the Retinoblastoma (RB) protein by the Cdk4/Cdk6 proteins in complex with Cyclin D.14 Phosphorylated RB dissociates from the E2F transcription factor and alleviates inhibition of E2F to allow transcriptional stimulation of Cyclin E and other S-phase promoting genes by E2F and immediate early transcription factors.14 Association of Cyclin E with Cdk2 leads to hyper-phosphorylation of RB, thus stimulating cell cycle progression beyond the G1/S checkpoint and committing the cell to enter S-phase. The CDK inhibitor (CDKI) proteins p21Cip1 and p27Kip1 negatively regulate CDK activity through protein complex formation, and phosphorylation of these CDKI proteins by Cdk2-Cyclin E is required to target these negative cell cycle regulators for degradation.14, 16Foxm1−/− regenerating hepatocytes display increased nuclear protein levels of the Cdk inhibitors (CDKI) protein p21Cip1 and p27Kip1, suggesting that Foxm1 controls the expression of genes that regulate nuclear levels of these CDKI proteins.35, 37, 38 Foxm1 is required for expression of S-phase promoting Cdc25A phosphatase,37 which dephosphorylates inhibitory Cdk2 residues and activates Cdk2-Cyclin E activity.14, 17 Foxm1 is essential for progression into mitosis because it regulates transcription of the Cdk1 activating Cdc25B and Cyclin B1 genes and the mitotic regulators Aurora B kinase, Survivin, and Polo-like kinase 1 genes.36-38, 40, 41 Survivin forms a complex with chromosome passenger proteins Aurora B kinase and Inner Centromere Protein (INCENP), where it plays a critical role in the localization of Aurora-B kinase-INCENP complex to the inner chromosomal region of centromeres at the early stages of mitosis.48 The cytokine (TNFα and IL-6) and growth factor (TGFα and HGF) induction of the Ras-MAPK signaling pathway is critical for activating immediate early transcription factors (AP1, C/EBPβ, NFκB and Stat3). These transcription factors regulate the temporal expression of Cyclin D1 regulatory subunit (Fig. 1), which activates its corresponding Cyclin-dependent kinase (Cdk) 4 and 6 through complex formation.13-15 Progression into DNA replication (S-phase) requires phosphorylation of the Retinoblastoma (RB) protein by the Cdk4/Cdk6 proteins in complex with Cyclin D (13) (Fig. 1). Phosphorylated RB dissociates from the E2F transcription factor and alleviates inhibition of E2F to allow transcriptional stimulation of Cyclin E and other S-phase promoting genes.15 Association of Cyclin E with Cdk2 leads to hyper-phosphorylation of RB, thus stimulating cell cycle progression beyond the G1/S checkpoint and committing the cell to progress into S-phase (Fig. 1). The CDK inhibitor (CDKI) proteins p21Cip1 and p27Kip1 also negatively regulate CDK activity through protein complex formation, and phosphorylation of these CDKI proteins by Cdk2-Cyclin E is required to target these negative cell cycle regulators for degradation14, 16 Progression from the G2 phase into mitosis requires expression of Cyclin B1 and activation of the Cdk1-Cyclin B complex through dephosphorylation and activation of Cdk1 by the Cdc25B and Cdc25C phosphatases14, 17 (Fig. 1). Expression of the Polo-like kinase (PLK1) and Aurora B kinase also occurs during G2 phase of the cell cycle and these kinases phosphorylate proteins that are critical for mitotic progression18, 19 (Fig. 1). Numerous genetic studies using either transgenic or knockout mice have demonstrated that activation of immediate early transcription factors is critical for transcriptional activation of the S-phase cyclin genes and expression of proteins involved in hepatocyte survival during PHx induced liver regeneration. These immediate early transcription factors include the c-Myc,20, 21C/EBPβ,6, 22 Crem-1,23 c-Jun24 and Stat325 genes. Expression of these transcription factors was required for normal levels of cell cycle regulatory Cyclins D1, E, or B1 proteins following PHx (Fig. 1), whereas the c-Jun is also necessary for hepatocyte survival during liver regeneration. These mouse genetic studies demonstrate that the induction of immediate early transcription factors is critical for hepatocyte DNA replication following PHx. CAR, constitutive androstane receptor; TNFα, tumor necrosis factor α; TGFα, tumor growth factor α; HGF, hepatocyte growth factor; IL-6, interleukin 6; AP1, activation protein 1 (c-Jun and c-Fos heterodimers); bHLH, basic-helix-loop-helix; bZIP, basic leucine zipper; C/EBPβ, CCAAT/enhancer binding protein β; NFκB, nuclear factor κB; Stat3, signal transducers and activators of transcription 3; MAPK, mitogen activated protein kinase; Cdk, cyclin-dependent kinase; CDKI, CDK inhibitor; RB, retinoblastoma protein; Foxm1, Forkhead Box m1; TCPOBOP, 1,4-bis-(2-(3,5,-dichloropyridyloxy)) benzene; INCENP, Inner Centromere Protein; LRF-1, liver regenerating factor 1. In contrast to liver regeneration induced by partial hepatectomy, primary mitogens induce hepatocyte proliferation without causing liver injury and the proliferative process induced by primary mitogens, direct hyperplasia, results in an increase of hepatic DNA and liver mass26 The synthetic ligand for the CAR nuclear receptor, TCPOBOP, produces a particularly strong and rapid proliferative response in mouse liver. The work by members of the Columbano laboratory, and their recent collaboration with the Locker laboratory, have contributed significantly to our understanding of the mechanisms involved in stimulation of hepatocyte proliferation in response to primary mitogens. They have previously shown that initiation of hepatocyte DNA in response to the CAR agonist TCPOBOP occurs much more rapidly compared to PHx induced liver regeneration, but that TCPOBOP treatment lacks many early events thought to be critical in liver regeneration.27 Activation of latent transcription factors (NFκB, STAT3), increased expression of immediate-early transcription factors (AP1, c-myc, LRF1, egr1), and increased synthesis of growth factors (TGFα, HGF) are all considered essential in liver regeneration, but none of these factors are induced during CAR− or other nuclear receptor-mediated hepatocyte proliferation28-30 (Fig 2). Consistent with earlier hepatocyte DNA replication, TCPOBOP treatment stimulated earlier expression of Cyclin D1 in proliferating hepatocytes (Fig. 2), suggesting that the CAR nuclear receptor directly activates transcription of Cyclin D1 without engaging the immediate early transcription factors.31, 32 Surprisingly, TCPOBOP treatment of Cyclin D1−/− mice exhibited a delay in hepatocyte proliferation, but it eventually stimulated hepatocyte proliferation at later time points through compensatory increase of the downstream Cyclin E protein33 (Fig. 2). Regulation of cell cycle genes in proliferating hepatocytes following CAR induced liver regeneration. Diagram of the cell cycle is described in Fig. 1 legend. CAR nuclear receptor bypasses induction of immediate early transcription factors and activates expression of Cyclin D128, 32, 49 and Foxm1 transcription factor,34 both of which are critical for progression into DNA replication and mitosis.14, 39 CAR nuclear receptor also stimulates expression of Gadd45β gene, which is involved in preventing apoptosis. Although both the TNFR1−/− mice and TNFR1−/−TNFR2−/− double knockout mice are nonresponsive to PHx induced liver regeneration,10, 12 Columbano and colleagues convincingly demonstrated that TCPOBOP treatment strongly stimulated hepatocyte proliferation, and at significantly higher levels than wildtype (WT), in the TNFR-deficient mice, and that this was associated with higher levels of Cyclin D1 protein.11 The greater induction of proliferation in TNFR-null mice suggests a negative cross talk between the TCPOBOB-CAR and TNFα-–NFκB signaling pathways that regulate hepatocyte proliferation. Furthermore, TCPOBOP induced hepatocyte proliferation in both WT and TNFR-deficient mice without detectable induction of the NFκB or Stat3 transcription factors,11 which are normally induced in regenerating liver following PHx.1, 2, 4 To examine whether the TNFα–NFκB and CAR signaling pathways are distinct, the authors chose to examine the expression of Gadd45β/MyD118 gene, a member of the Gadd45 family of inducible factors, which is induced by both partial hepatectomy and TCPOBOP treatment. They showed that TCPOBOP induced hepatic expression of Gadd45β in both WT and TNFR-deficient mice equally, and therefore the lack of NFκB activation did not impair CAR-mediated Gadd45β gene induction.11 Further studies evaluated TCPOBOP-induced gene expression in CAR−/− mice by microarray expression profiling and Northern blot. The induced changes in gene expression, including the stimulation of Gadd45β, were almost completely abolished—hence all were mediated via activation of the CAR nuclear receptor.11 The results suggest that, in the liver, Gadd45β is induced by a distinct pathway that requires CAR and is independent of TNFα–NFκB signaling pathway. Previous studies from Dr. Columbano's laboratory demonstrated that treatment of old-aged (12-month-old) mice with the CAR agonist TCPOBOP caused an increase in hepatocyte DNA replication and mitosis similar to that seen in young (2-month-old) mice.34 Stimulation of hepatocyte DNA synthesis by TCPOBOB was associated with increased expression of several cell cycle-associated proteins (cyclin D1, cyclin A, cyclin B1, E2F, Rb, and p107); all of which exhibited comparable expression in old-aged mice and young mice. TCPOBOP treatment was also shown to increase expression of the Forkhead Box m1 (Foxm1 or FoxM1b previously known as HFH-11B) transcription factor in young mice and restore induced levels of Foxm1 mRNA in old-aged mice.34 Foxm1 is known to regulate transcription of genes required for regenerating hepatocyte progression into DNA replication and mitosis35-38 and induced expression of Foxm1 is severely compromised during liver regeneration in old-aged mice.39-41 Furthermore, maintaining expression of Foxm1 alone in hepatocytes of old-aged mice was sufficient to restore hepatocyte DNA synthesis, mitosis and expression of cell cycle regulatory genes to levels found in young regenerating mouse liver39-41 (Fig. 1). Induction of Foxm1 expression by TCPOBOP in old aged mice is thus consistent with normal levels of hepatocyte proliferation and reestablishing expression of cell cycle regulatory genes (Fig. 2). These data suggest that Foxm1 is an important target of the TCPOBOP-CAR signaling pathway in stimulating hepatocyte proliferation in old-aged mice. During mouse liver regeneration, Foxm1 expression is induced in mid-G1 phase of the cell cycle and premature expression of Foxm1 (HFH-11B) caused earlier onset of hepatocyte DNA replication and mitosis and earlier expression of the appropriate cell cycle gene required for entry into S-phase and mitosis.42, 43 Liver regeneration studies with mice in which the Albumin promoter-enhancer Cre Recombinase transgene (Alb-Cre) mediated conditional deletion of the Foxm1 LoxP/LoxP (fl/fl) targeted allele in adult hepatocytes demonstrated that Foxm1 is required for high levels of regenerating hepatocyte DNA replication and is essential for mitosis.37, 39 These studies demonstrated that Foxm1 is essential for normal expression of Cdc25A phosphatase and diminishing nuclear accumulation of CDK inhibitor (CDKI) proteins p21Cip1 (p21) and p27Kip1, all of which are required for progression into S-phase35, 37, 39 (Fig. 1). For progression into mitosis, Foxm1 regulates transcription of the Cdk1 activating Cdc25B phosphatase and Cyclin B1 (Fig. 1) and the mitotic regulators Aurora B kinase, Survivin and Polo Like Kinase 1, which phosphorylate substrates essential for executing mitosis35-38, 40 (Fig. 1). Reduced cellular proliferation during aging is associated with a progressive decline in both growth hormone (GH) secretion and Foxm1 expression.40, 44, 45 Human GH administration to old-aged mice dramatically increased expression of Foxm1 and its cell cycle target genes and restored proliferation of regenerating hepatocytes to levels found in young regenerating liver46, 47(Fig. 1). Furthermore, the TCPOBOP agonist of the CAR nuclear receptor strongly induces hepatocyte proliferation in either old aged mice or TNFR1-deficient mice, both of which are normally nonresponsive to surgically induced liver regeneration. Altogether, these studies show that both growth hormone and TCPOBOP are capable of stimulating suboptimal old-aged livers to proliferate and undergo regeneration. Based on these studies, either CAR agonist or growth hormone is potentially useful to stimulate hepatocyte proliferation during the liver regeneration period following liver transplantation of suboptimal cadaver donor livers. These hepatic mitogens may also be effective for short-term use to enhance the liver regeneration process in live-donor transplants for both recipient and donor patients.