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Abstract

Background & Aims: Under favorable microenvironment, nuclear receptor FXR controls apical ABC-transporter BSEP/ABCB11 for bile acid delivery. In acute-on-chronic liver failure (ACLF), most patients lack nuclear FXR in hepatocytes, but maintain apical BSEP expression. The current study investigated the mechanisms of how hepatocytes maintain BSEP expression at the absent of FXR.

Methods: We observed liver tissues from 3 controlled, 5 chronic HBV infected, and 18 ACLF patients. Among ACLF patients, 13 received liver transplantation and 5 recovered. BSEP and target transcription factors were examined by immunohistochemistry in the collected liver tissues. BSEP regulatory mechanisms were investigated in AML12 cell line, mouse primary hepatocytes, human primary hepatocytes and fxr knockout mice.

Results: Firstly, we used immunohistochemical staining (IHC) to examine BSEP expression in 26 liver tissues. IHC analysis showed intact BSEP apical expression in controlled, chronic HBV infected and recovered ACLF patients. In irreversible ACLF patients, 10 displayed BSEP expression in most hepatocytes while 3 did not have detectable BSEP expression in most areas of the specimens. Fifteen ACLF patients who maintained BSEP expression did not have detectable nuclear FXR expression. However, they expressed robust nuclear FOXA2 in hepatocytes. We further compared clinical parameters between ACLF patients possessing and lacking BSEP expression. The recovered patients showed remarkably improved serum total bilirubin, international normalized ratio and Model for End-stage Liver Disease score than those receiving liver transplantation (p<0.01 for all parameters). In addition, compared to the irreversible patients possessing BSEP, the patients who lost apical BSEP demonstrated higher serum total bilirubin concentration (p<0.05). These results suggest that the maintenance of BSEP in hepatocytes is important for the recovery of ACLF. In vitro, overexpression or knockdown FOXA2 induced or inhibited BSEP expression in hepatocytes. Furthermore, ectopic FOXA2 expression restored apical BSEP expression in hepatocytes of fxr knockout mice in vitro and in vivo. ChIP assay revealed that both FXR and FOXA2 initiated ABCB11/BSEP transcription through binding to the gene promoter. However, administration of FXR agonist reduced FOXA2 binding to the ABCB11/BSEP gene promoter, indicating a competitive effect between FOXA2 and FXR on binding to the ABCB11/bsep gene promoter. In normal hepatocytes, insulin impeded FOXA2 nuclear translocation while glucagon induced FOXA2 expression. Under inflammatory condition, inflammatory factors, e.g. LPS and TNFalpha, resulted in high levels of glucagon and inhibition of FXR expression. Further, TNFalpha induced hepatocyte insulin resistance and thus upregulated FOXA2-dependent BSEP expression. In addition, LPS exploited NFκB signaling and thus initiated foxa2 transcription through p65 binding to the promoter. Notably, high concentration of LPS led to FOXA2 nuclear exclusion through phosphorylating at Thr156 of FOXA2, which reduced apical BSEP expression in hepatocytes.

Conclusions: The findings of this study illustrate two regulatory mechanisms relevant for the maintenance of BSEP on bile canaliculi in physiological and pathological conditions. (1) In physiological condition, FXR regulates BSEP expression. (2) In inflammatory circumstance, FXR is significantly inhibited. Inflammation-dependent insulin resistance, high levels of glucagon and NFκB p65 signaling induce FOXA2 expression and activity to maintain BSEP expression, which is important for ACLF patients’ survival and recovery. (3) In sepsis condition, severe infection and inflammation, e.g. high levels of LPS, inhibit FOXA2 activation and thus lead to the loss of BSEP expression on bile canaliculi, which are associated with poor prognosis of ACLF patients. In conclusion, the maintenance of BSEP is essential for survival and recovery of ALCF patients.