STUDY OF FXR IN PRIMARY HUMAN HEPATOCYTES AND FXR REGULATED BA HOMEOSTASIS IN PARENTERAL NUTRITION ASSOCIATED LIVER DISEASES

Farnesoid X receptor (FXR, NR1H4) is a ligand activated transcription factor belonging to the nuclear receptor (NR) superfamily, and is highly expressed in the liver, intestine, and kidney, in both humans and rodents. Bile acids (BAs) are the endogenous ligands of FXR. FXR mainly functions as the BA sensor by regulating genes that are critically involved in BA homeostasis. FXR has also been shown to play important roles in lipid, cholesterol and glucose metabolism, as well as inflammation, tumorigenesis, and liver regeneration. FXR deficiency is implicated in numerous liver diseases and mice with modulation of FXR have been used as animal models to study liver physiology and pathology. Genome-wide studies in mouse livers and intestines suggest FXR's diverse and broadly tissue specific functions. In the first aim, we studied the genome-wide FXR binding and transcriptome profiles upon FXR activation in primary human hepatocytes (PHHs) and HepG2 cells. Chromatin immunoprecipitation followed by massive parallel sequencing (ChIP-seq) was performed in PHHs and HepG2 cells, treated with a synthetic FXR agonist, GW4064 or DMSO control. In parallel, RNA deep sequencing (RNA-seq) and RNA microarray were performed for GW4064 or control treated PHHs and wild type (WT) mouse livers, respectively. ChIP-seq showed similar profiles of genome-wide FXR binding in humans and mice in terms of motif analysis and pathway prediction. However, RNA-seq and microarray showed more different transcriptome profiles between PHHs and mouse livers upon GW4064 treatment. In summary, we have established genome-wide human FXR binding and transcriptome profiles. These results will aid in determining the human FXR functions, as well as judging to what level the mouse models could be used to study human FXR functions in future studies. Recent studies in the field of parenteral nutrition (PN) suggest that down-regulation of FXR signaling is critically involved in the pathogenesis of PN associated liver diseases (PNALD), especially PN associated cholestasis (PNAC) in preterm infants. PN is a life-saving therapy for patients who cannot tolerate enteral food intake. However, long-term PN can lead to a spectrum of liver diseases, summarized as PNALD. PNAC is predominately found in preterm infants and neonates, and has high rate of progression into liver failure. Options for clinical management of PNALD are still limited. While it is suggested that multiple risk factors are contributing to the development of PNALD, the pathogenesis of PNALD remains poorly understood. Animal studies obtained from preterm piglets and several mouse models have shed light on the mechanisms underlined. To date, whether FXR and BAs are involved in the development and progression of PNALD is not well studied. In the second aim, we established a valid mouse PN model in our laboratory. Using RNA microarray profiling and serum BA profiling, we identified novel signatures involved in BA homeostasis in adult PN mice. We detected significantly increased gene expression of cytochrome P450, family 7, subfamily a, polypeptide 1 (Cyp7a1) and decreased gene expression of cytochrome P450, family 8, subfamily b, polypeptide 1 (Cyp8b1) in the livers of PN mice. Several FXR and liver X receptor alpha (LXRα) target genes involved in BA homeostasis were also altered in the livers of PN mice. Consistent with the gene expression alterations, both the levels and the percentages of tauro β-muricholic acid (T-β-MCA) as well as total non-12α-OH BAs in the serum of PN mice increased significantly compared to saline controls. These results suggest that BA homeostasis could be disrupted in PN patients as well and the deregulation of BA synthesis and metabolism could contribute to the development and progression of PNALD when additional risk factors are present. Additional altered genes and pathways were also detected from the microarray analysis for the PN mice. These results will aid us in future studies of the contributions of FXR and BA signaling, as well as other potential factors to the development and future management of PNALD