Guarding the gate against hyperbilirubinaemia

Hyperbilirubinaemia presents as jaundice, occurs frequently in newborn babies, and is a leading cause of hospitalization in the first weeks of life. In some infants, jaundice can progress to acute bilirubin encephalopathy and kernicterus, which may lead to neonatal mortality and neurodevelopmental impairments ¹. The degree of hyperbilirubinaemia is one indicator of the severity of liver failure. High levels of bilirubin in plasma may predict short-term mortality in these patients ². Four distinct steps of bilirubin metabolism were proposed ³. In health, unconjugated bilirubin is taken up along the sinusoidal surface of the hepatocyte by solute carrier family 21, member 6 (SLC21A6; AKA organic anion transporter 2, OATP2). Ligandin, a homodimer or heterodimer of glutathione-S-transferase (GST) A1 and A2, binds bilirubin with high affinity and increases its uptake. Bilirubin is then glucuronidated by bilirubin uridine diphosphate-5′-glucuronosyltransferase (UDP-glucuronosyltransferase 1A1, UGT1A1). The resulting hydrophilic bilirubin diglucuronide is then secreted across the bile-canalicular membrane of the hepatocytes by multidrug resistance–related protein 2 (MRP2) (cMOAT, ABCC2). Disrupting the pathway anywhere in this process can lead to several hyperbilirubinaemia syndromes. For example, complete OATP1B1 and OATP1B3 deficiency interrupts unconjugated bilirubin reuptake into the liver and leads to Rotor syndrome ⁴. Mild to severe deficiency of UGT1A1 results in two types of familial unconjugated hyperbilirubinaemia, Crigler-Najjar syndromes I and II, and Gilbert’s syndrome ⁵. Dubin–Johnson syndrome, a heritable loss of human MRP2 function, is characterized by hyperbilirubinaemia and pigment deposition in the liver. MRP2 is the rate-limiting step in hepatic bilirubin excretion. Ligands for farnesoid X-activated receptor (FXR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR), such as chenodeoxycholic acid, PCN, dexamethasone, and phenobarbital, induced MRP2 mRNA in rat hepatocytes ⁶. Data supported a putative ER-8 at 401 to 376 of the rat MRP2 promoter with high affinity upon for the retinoid X receptor (RXR) ⁶. However, in disease, the regulatory mechanism of MRP2 transcription is largely unknown.

In this issue, Wang and colleagues show that the transcription factor Forkhead box A2 (FOXA2) may be a surrogate for FXR and regulate MPR2 expression in inflammation. Confirming a prior report ⁶, the authors found that FXR^−/− mice lacked MRP2 expression and were hyperbilirubinaemia, suggesting that FXR is crucial for MRP2 expression and bilirubin delivery. However, in many individuals with acute liver failure (ALF), FXR, PXR, CAR and RXR were undetectable in the nuclei of hepatocytes although some of these individuals still demonstrated intact MRP2 expression in hepatocytes. These findings suggest the existence of a compensatory mechanism that maintains MRP2 transcription in the absence of FXR, PXR, CAR and RXR. The authors found that FOXA2 was undetectable in individuals without ALF. In contrast, hepatocyte FOXA2 was strongly expressed in ALF patients. As well, FOXA2 upregulated MRP2 transcription through binding to its promoter. In vivo experiments confirmed the effect of Ad-FOXA2 on hepatocyte MRP2 expression to limit hyperbilirubinaemia in FXR^−/− mice. Relevant to this, hepatic FOXA2 mutant mice displayed decreased MRP2 expression ⁷. These findings suggest that FOXA2 is an alternative transcription factor that helps maintain hepatic ABCC2/MRP2 transcription such as in ALF when FXR and other NRs are inactivated.

In addition, the authors found that TNFα increased hepatocyte FOXA2 and MRP2 expression while inhibiting FXR. Specifically, TNF-α increased FOXA2 binding to the Mrp2 promoter, suggesting that TNFα could upregulate FOXA2 expression and nuclear translocation to maintain hepatocyte apical MRP2 expression in situations of severe liver damage.

Not unexpectedly, septic individuals with ALF had more severe hyperbilirubinaemia. However, hepatocyte FOXA2 and MRP2 were not detectable in the septic individuals. In line with this, LPS increased phosphorylated and total FOXA2 expression and inhibited FXR and MRP2, suggesting that sepsis may interfere with FOXA2-mediated compensatory MRP2 upregulation. Finally, forced FOXA2 expression restored canalicular MRP2 expression and attenuated hyperbilirubinaemia in LPS-treated mice. Whether this would be a means of treating hyperbilirubinaemia in individuals with ALF remains to be tested (Fig. 1).

Figure 1.

Nuclear receptors (NR) and FOXA2 regulate hepatocyte apical MRP2 expression under different pathophysiological conditions. MRP2 is an apical transporter that excretes conjugated bilirubin from hepatocytes into bile canaliculi. In health, NRs (FXR, CAR, PXR, RXR) control apical MRP2 expression. Under inflammation, TNF-a inhibits the expression of FXR and other NRs, but induces FOXA2 expression. FOXA2 upregulates MRP2 transcription through binding to its promoter. Similarly, LPS phosphorylates FOXA2 to induce FOXA2 nuclear exclusion. Both NRs and FOXA2 binding to the MRP2 promoter is prohibited. Loss of MRP2 expression results in hyperbilirubinaemia. BG, bilirubin glucuronide; UCB, unconjugated bilirubin.

Besides delivering bilirubin into bile, MRP2 is also an apical efflux pump for secretion of bile aids (BAs). BAs are oxidized derivatives of cholesterol produced by the liver to facilitate absorption of dietary lipids. Dysregulation of BA homeostasis can cause cholestatic liver disease. MRP2 deficient rats demonstrated higher plasma BA concentrations consistent with reduced BA biliary secretion and increased BA efflux from hepatocytes to blood via upregulated multidrug resistance-associated protein 3 (Mrp3) and multidrug resistance-associated protein 4 (Mrp4) transporters ⁸. The animals had increased plasma deoxycholic acid (DCA) compared to controls, which in turn raised the 12α-hydroxylated/non-12α-hydroxylated BA ratio. These data suggest that, under MRP2 deficiency, there is a change in net BA transport and synthesis of secondary BAs. It appears that FOXA2 is also required for normal BA homeostasis. FOXA2 expression was severely reduced in livers from individuals with various cholestatic syndromes ⁷. Moreover, FOXA2 mutant mice had accumulation of hepatic BAs ⁷. Analysis of the transcription of levels of genes encoding BA transporters, including Mrp2, Mrp3, Mrp4 and Oatp2, would be warranted. The novel findings in this study may have implications beyond hyperbilirubinaemia, including cholestasis due to BA accumulation in the liver. Another potential interesting question is whether the same mechanism applies to other apical transporters, for example the bile salt export pump (BSEP), a rate-limiting transporter for BAs.

Among NRs known to regulate Mrp2 expression, CAR and PXR more specifically regulate liver responses to xenobiotics and endobiotics, including drugs, environmental chemicals, bilirubin and bile acids. Previous reports identified CAR as a key regulator of bilirubin detoxification and clearance in the liver ^{9. 10}. In response to elevated bilirubin levels, CAR activates the expression of essentially all components of the bilirubin clearance pathway, including OATP, GSTA, UGT1A1 and MRP2, which results in increased bilirubin clearance. Yin Zhi Huang, an herb decoction widely used in Asia to prevent and treat neonatal jaundice, works through CAR activation to accelerate bilirubin clearance. In response to inflammation, CAR expression was also decreased in FXR^−/− livers. This raises the possibility that the physiological role of CAR in regulating Mrp2 may be underestimated in this study. Alternatively, one may posit that all NRs (FXR, CAR, PXR, RXR) work individually to regulate Mrp2 expression. ALF suppresses all NRs-dependent Mrp2 regulation. Thus, FOXA2 represents an alternative mechanism to maintain Mrp2 expression (Fig. 1). It would be interesting to know whether FOXA2 also regulates OATP, UGT1A1 and other components of bilirubin metabolism in ALF.

In conclusion, the current studies add FOXA2 as an alternative guard of NRs to limit hyperbilirubinaemia. In septic individuals with ALF, induction of hepatic FOXA2 may provide relief from hyperbilirubinaemia.