| Part I:Bile acid elevates estrogen levels via FXR-mediated suppression of SULT1E1 during cholestasisLiver is the main site of peripheral estrogen inactivation and catabolism. Liver disease is usually associated with the abnormal estrogen status through an unknown mechanism. Bile acids (BA) are important liver products and substantially increase in cholestatic liver disease. BA are endogenous ligands for the Famesoid X receptor (FXR). FXR is a nuclear receptor highly expressed in the liver. It regulates genes involved in bile acid synthesis, lipid and lipoprotein metabolism, including small heterodimer partner (SHP), cholesterol 7α-hydroxylase (CYP7A1), sterol-12a-hydroxylase (CYP8bl), bile salt export pump (BSEP), Apolipoprotein (Apo) AI, Apo CⅡ, Apo CⅢ and the phospholipid transfer protein (PLTP). In addition, FXR also plays a crucial role in glucose metabolism, insulin sensitivity, and atherosclerosis. FXR has been extensively studied as a new therapeutic target in numerous metabolic disorders. However, the effect of bile acid activated FXR on estrogen metabolism in liver remains unknown.We consistently noticed that patients suffering from obstructive cholestasis exhibited clinically very high levels of estrogen. We conducted a systematic analysis of these patients for markers of cholestasis, such as bilirubin, y-GT, and serum bile acid concentrations. The results were correlated with estrogen levels. We next investigated the effect of BA on E2 levels in patients with PBC. Forty postmenopausal female patients with PBC were divided in two groups:patients with normal serum BA levels (low BA group, n=22) and patients with high serum BA levels (high BA group, n=14). We found that high BA group had significantly elevated E2 levels (454.5±151.8 pmol/L) compared with low BA group (91 ± 41.5 pmol/L). After appropriate treatment, the BA concentrations were normalized in 8 patients from high BA group. Interestingly, serum E2 level in those 8 patients dropped from (492.9 ± 159 pmol/L) to (185.5 ± 86.5 pmol/L). Moreover, serum E2 was positively correlated with serum BA levels in high BA group (R2=0.317, P= 0.0361). Overall, these data suggested that BA might increase E2 levels.FXR deficiency causes cholestasis. To investigate the role of FXR in regulating E2 metabolism in vivo, we first studied the BA and E2 concentrations in WT and FXR-/- female mice. Compared with WT mice, FXR-/- mice showed elevated serum BA, which was consistent with previous report. Surprisingly, decreased E2 level was observed in FXR-/- mice. We next profiled hepatic expression of key genes involved in estrogen metabolism. Among 8 primary estrogen-metabolizing enzymes, the transcript level of SULT1E1 in FXR-/- mice was increased dramatically by about 500-fold compared with WT mice. Aside for a slightly higher expression of HSD17B1 and STS in FXR-/- mice, no difference in hepatic expression of UGT1A1, CYP1A1, CYP1A2, CYP1B1 and CYP19A1 between FXR-/- and WT mice was observed. The observed decrease expression of SHP, a known FXR target gene, in FXR-/- mice confirmed FXR deficiency. Immunohistochemistry and Western blotting confirmed that the protein levels of SULT1E1 were increased in FXR-’-mice compared with WT mice. Interestingly, no difference in protein levels of HSD17B1 and STS between FXR-/- and WT mice was observed. We subsequently examined SULT1E1 protein levels in a panel of tissues from female mice. SULT1E1 protein levels were significantly increased in the kidney, liver and colon of FXR-/-mice compared with WT mice. SULT1E1, a key enzyme in estrogen metabolism, is responsible for the inactivation and elimination of E2 at physiological concentrations. Therefore, we measured the clearance of exogenously administrated E2 in female mice. At 6 h after E2 administration, the E2 levels in FXR-’-mice were significantly lower than the WT mice, while at 12 h no difference was observed.We studied the influence of FXR agonists on SULT1E1 expression in human liver Huh7 cells. Real-time quantitative PCR and Western blotting revealed a significant decrease in both transcript and protein levels of SULT1E1 after FXR agonist’s treatment. In contrast, FXR knockdown in Huh7 cells led to a significant increase of SULT1E1 mRNA and protein levels. Co-treatment with CHX did not block WAY-362450-induced suppression of SULT1E1 expression, thus suggested that protein synthesis was not required for FXR-mediated repression of the SULT1E1 gene.We next searched for the putative FXR-binding sites in the intragenic regions upstream of the transcriptional start site of SULT1E1 gene in the human and mouse genome. No conservative FXR response element was present in the SULT1E1 promoter. We then searched for the occurrence of putative FXR response elements of the SULT1E1 promoter using the genome wide ChIP-seq datasets published by Thomas et al and Boergesen et al. The FXR UCSC genome browser tracks did not show any FXR-binding signal in the intragenic regions upstream of the transcriptional start site of SULT1E1 gene. Kodama et al found that knockdown of hepatocyte nuclear factor 4a (HNF4a) reduced the SULT1E1 mRNA levels by 90%. Interestingly, we found that FXR strongly repressed HNF4a-stimulated SULT1E1 promoter activity in a dose-dependent manner. Surprisingly, qChIP assay demonstrated the occupancies of FXR were not changed at the SULT1E1 promoter after treatment with WAY-362450. However, agonist-activated FXR remarkably reduced the binding of HNF4a to SULT1E1 promoter. Moreover, activated FXR enriched the H3K27me3 repressive mark in the SULT1E1 promoter. In contrast, FXR knockdown augmented the binding of HNF4a to SULT1E1 promoter and removed the H3K27me3 repressive mark in the SULT1E1 promoter. The repression function of agonist-activated FXR on SULT1E1 promoter activity was abolished by deletion of the direct repeat half sites (DRs), which composed of three direct repeats of the motif GGACC and referred to as an HNF4a response element. Taken together, we reported that the elevated BA increased the E2 levels via FXR activation during cholestasis. These findings suggest a novel role for bile acid activated FXR on hepatic metabolism of estrogen during cholestasis.Part II:Farnesoid X receptor functions as a tumor suppressor through antagonizing Wnt/β-Catenin signaling and is frequently silenced in hepatocellular carcinomaFarnesoid X receptor (FXR) participate in the regulation of multiple physiological activities, including bile acid, lipid and glucose metabolism. Oncogenic activation of the Wnt/β-Catenin signaling pathway is closely associated with human hepatocellular carcinoma (HCC). Previous studies have shown that FXR deficiency led to spontaneous hepatocarcinogenesis and dysreguation of Wnt/β-Catenin signaling, but the molecular mechanisms still remain unknown. Here, we report the mechanisms by which FXR cross-talks with Wnt/β-Catenin signaling to regulate HCC tumorgenesis. Silencing expression of FXR and aberrant activation of β-Catenin in the human HCC cell are negatively correlated. To determine whether FXR involved in Wnt/β-Catenin signaling, we performed a Western blot analysis for FXR, total and active β-Catenin in several hepatocyte carcinoma cell lines. Interestingly, the Western blot analysis indicates an increase in active β-Catenin but a decrease in FXR.Next, we investigate whether FXR interact with β-Catenin which plays a key mediator in Wnt signaling pathway. Endogenous FXR bound to β-Catenin in PLC-5 cells, as determined by coimmunoprecipitation assay. The interaction between FXR and P-Catenin was confirmed by immunoprecipitation assay of HEK293T cells transfected with Myc-FXR and HA-P-Catenin. The direct interaction between FXR and β-Catenin was further analyzed in vitro using recombinant GST-β-Catenin and His-FXR. In a GST pull-down assay, the purified GST-β-Catenin bound to His-FXR but not GST alone. Moreover, we examined the cellular localizations of FXR and β-Catenin using confocal microscopy. The merged images indicated that FXR and β-Catenin proteins were colocalized in both cytoplasm and nuclear. To further investigate the as-yet-unidentified functional modules of FXR, we prepared a serious FXR truncation, co-IP assay indicated that the N-terminal domain (NTD including AF1, DBD and Hinge domains) of FXR is dispensable for β-Catenin binding. To elucidate the consequences of the interaction of FXR with β-Catenin, we investigated whether FXR directly inhibits the function of β-Catenin. FXR and β-Catenin were cotransfected into HEK293T cells together with the TOPflash reporter plasmid or control plasmids. The transfected FXR strongly inhibits the P-Catenin-mediated luciferase activity driven from the TOPflash reporter in a dose dependent manner, while FXR had no effect on the control FOPflash reporter plasmid.Since our previous results showed that FXR can negatively regulate the Wnt/β-Catenin signaling through interaction with β-Catenin, we next determined whether the other core transcription factors-TCF4 interacts with FXR and FXR affects the assembly of the β-Catenin-TCF4 transcription activation complex. Surprisingly, we found that exogenously expressed Myc-FXR and HA-TCF4 did not form a ternary complex. We investigated whether gain or loss of function of FXR disturbs the stability of β-Catenin-TCF4 complex. We found that the binding between P-Catenin and TCF4 was decreased upon FXR activation in Wnt3a-treated Huh7 cells. Moreover, the association of P-Catenin-TCF4 complex with the Cyclin D1 promoter upon FXR activation by GW4064 was decreased. In contrast, when FXR was deleted in Huh7 cells by two independent siRNA, the interaction between P-Catenin and TCF4 was enhanced and the binding of β-Catenin-TCF4 to Cyclin D1 promoter was increased. ChIP assays revealed that gain or loss of function of FXR can greatly affect the recruitment of β-Catenin-TCF4 complex on the Cyclin D1 promoter. These results further strengthen our conclusion that FXR represses Wnt activity by disrupting the P-Catenin-TCF4 complex and inhibition of the binding of β-Catenin-TCF4 to Wnt response elements (WREs). Taken together, we identify FXR as a binding partner of β-Catenin and FXR negatively regulates the transcriptional activity of Wnt signaling by disrupting the assembly of the core β-Catenin/TCF4 complex. Activation of FXR attenuates the DNA-binding activity of P-Catenin/TCF4. The ectopic expression of FXR causes strong inhibition of cell growth, migration and invasion in vitro and nude mouse xenografts in vivo. By contrast, knockdown of FXR using shRNA provokes Wnt3a induced the luciferase activity of TOPflash and exaggerates the proliferation and tumorgenesis in HCC cells, whereas downregulation of P-Catenin rescues the enhanced effect. Taken together, the elucidation of the mechanism how FXR control the proliferation and tumorgenesis of hepatocellular carcinoma cells is helpful for the liver cancer treatment. These findings show that FXR exerts inhibitory effect on Wnt/β-Catenin signaling mediated tumorgenesis in HCC cells. |
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