CAR takes care of the injured liver

In contrast to other organs, which heal with scar formation, the liver has the unique capability to fully regenerate after injury [, ]. This remarkable regenerative potential is of major importance after resection of liver tissue in patients with primary or metastatic liver tumors, in liver transplant patients, who usually receive small liver grafts that require expansion of the transplanted liver mass, or after liver cell loss caused by viruses, autoimmune diseases and toxins. These include alcohol or commonly used anti-inflammatory, anticonvulsant, or chemotherapeutic drugs. Rapid and effective liver repair is essential due to the critical and indispensable function of this organ in the regulation of metabolism and detoxification of endogenous and exogenous compounds. However, the regenerative capacity is insufficient when the remaining liver volume is too small. In the mouse, this is the case when more than approximately 85% of the liver is removed. Under these conditions, rapid liver failure occurs and the mice die within 48 h []. The requirement of a critical liver volume for efficient regeneration also limits the application of liver surgery in humans, for example in patients with liver cancer, where a large part of the organ has to be surgically excised. Similarly, liver transplantation frequently fails when the transplanted liver mass is too small. The resulting liver failure is known as small-for-size syndrome (SFSS), and it is characterized by liver dysfunction and severe hepatosteatosis, ultimately leading to the death of the affected patient within a few days after surgery. Unfortunately, there are as yet no therapeutic options for the treatment of SFSS, which is at least in part due to the insufficient understanding of the underlying pathomechanisms.

A recent study suggested that SFSS is not predominantly the result of the massive injury, since SFSS also occurred in a mouse model of 86% hepatectomy that induced little injury []. Rather, the failure of the hepatocytes in the remaining liver to progress through the cell cycle was shown to result in impaired regeneration and ultimately liver failure []. Therefore, there is a strong need to develop strategies to overcome this block in cell cycle progression and regeneration. In a recent study, loss of the cell cycle inhibitor p21 rescued mice from SFSS after 86% hepatectomy [], but such an approach is not a therapeutic option in patients. Rather, application of pharmacological compounds, which promote cell cycle progression and also improve the metabolic activity of the remaining liver tissue, would be a promising therapeutic strategy. In this issue of the Journal of Hepatology, Tschuor et al. propose the use of agonists for the constitutive androstane receptor (CAR; Nuclear receptor subfamily 1, group I, member 3; Nr1i3) for the improvement of liver regeneration []. CAR is a member of the nuclear receptor family with an important role in detoxification of endo- and xenobiotics. It is not only activated by several endogenous ligands, including the steroid hormone androstenol and isomers thereof, and by bilirubin but also by exogenous compounds. These include TCP (1,4,-bis(2-(3,5-dichloropyridyloxy))benzene, which activates mouse Car, and CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime), which activates human CAR. In addition, the anticonvulsant phenobarbital and some derivatives can indirectly activate CAR by inducing its dephosphorylation and subsequent nuclear accumulation []. Activation of CAR results in upregulation of genes encoding certain members of the cytochrome P450 enzyme family as well as sulfotransferases and glutathione-S-transferases, thereby allowing efficient compound detoxification and secretion []. In addition to these metabolic functions, a potent role of CAR in liver cell proliferation has been discovered. Thus, Car activation in mice via phenobarbital-like compounds rapidly induces reversible hepatomegaly through induction of a replicative response [, ]. This involves activation of c-Myc and and its downstream target, forkhead transcription factor M1 (Foxm1), a major regulator of several cyclins []. Through its dual activities in the regulation of metabolism and hepatocyte proliferation and its previously demonstrated cytoprotective function in the liver [], activation of CAR is an excellent candidate approach for the improvement of liver regeneration, and this possibility was tested by Tschuor et al. []. In support for an important role of CAR in liver regeneration, they first showed that the upregulation and activation of Car seen after 70% hepatectomy in mice is strongly impaired after 86% hepatectomy. Most importantly, Car deficiency resulted in the development of SFSS (Fig. 1) after 70% hepatectomy, which was reflected by reduced survival, hepatosteatosis, liver dysfunction and reduced hepatocyte proliferation that was associated with a reduction in Foxm1 and Foxm1-regulated cyclins, but upregulation of the cell cycle inhibitor p21. Thus, Car knockout mice revealed a similar phenotype after 70% hepatectomy as wild-type mice after 86% hepatectomy. This severe phenotype seems to be contradictory to the mild abnormalities in regeneration after partial hepatectomy that were previously reported for Car-deficient mice []. However, the survival rate and the rate of hepatocyte proliferation are not mentioned in this publication and therefore, the severity of the liver regeneration defect is not fully clear.

Even more important was the finding that activation of Car by oral gavage application of TCP during 86% hepatectomy suppressed p21, elevated the proliferation rate of hepatocytes and normalized the metabolic features seen after extended surgery. Remarkably, it even allowed survival of 40% of the mice with 91% hepatectomy or after transplantation of 30% (v/v) of the liver. Both procedures normally result in 100% lethality. Thus, Car activation in mice was indeed able to protect the animals from the development of SFSS (Fig. 1). This was even observed when TCP treatment was performed after surgery, suggesting a therapeutic window of Car activation and thus its use for treatment and not only for prevention of SFSS. Using a recently developed nanoparticle-based approach for selective gene knockdown in hepatocytes during liver regeneration [], Tschuor and colleagues further showed that Foxm1 mediates the effects of Car in their experimental SFSS model, since Car activation was ineffective after Foxm1 knockdown. The human relevance of the findings was addressed using mice expressing a humanized version of CAR instead of the mouse protein. Activation of CAR by CITCO also had a beneficial effect on liver regeneration in these mice, although the effect was less pronounced compared to wild-type mice treated with TCP, most likely due to the lower CAR activation capability of CITCO compared to TCP. In addition, treatment of ex vivo cultures of human liver slices improved the histological appearance, induced a proliferative response in hepatocytes, and enhanced liver cell viability at high doses. These findings suggest CAR activation as a potential strategy for the treatment of SFSS in humans, although this will most likely require novel and more potent ligands for human CAR. However, even if such ligands become available, a major concern remains, namely the potential pro-tumorigenic effect of CAR activation. This is particularly worrisome, since extensive hepatectomy is most often performed in patients with existing liver cancer and may thus promote tumor recurrence in these individuals. Consistent with this possibility, Car ligands promoted liver tumorigenesis in mice [], and the induction of hepatocarcinogenesis by chronic xenobiotic stress in mice was shown to be Car dependent []. This is particularly relevant in the presence of activated β-catenin, since it was recently shown that activation of both Car and β-catenin is required for the development of hepatocellular carcinoma (HCC) in mice. Furthermore, combined activation of CAR-mediated gene expression and of β-catenin was identified as a characteristic feature in a subset of human HCCs []. These findings suggest that further activation of CAR in patients with HCCs bearing activating mutations in the β-catenin gene should be avoided. Tschuor et al. further proposed to restrict future trials with CAR activators for the treatment of SFSS to patients showing downregulation of CAR in the tumor tissue. Activation of CAR for SFSS treatment is further encouraged by the lack of a clear association between phenobarbital treatment and liver carcinogenesis [], although further studies and larger patient numbers are required to further determine a possible pro-tumorigenic effect of this drug in the liver. Most importantly, induction of liver tumorigenesis by Car agonists was so far only observed in a chronic setting, whereas the mitogenic effect of acute Car activation on liver cell proliferation was reversible []. Thus, given the frequently fatal outcome of SFSS after liver surgery, a single treatment with CAR agonists for the prevention and/or treatment of this symptom is an exciting and well-justified perspective, in particular in appropriately selected patients.