Farnesoid X Receptor Knockdown Research Articles

Tauroursodeoxycholic acid suppresses biliary epithelial cell apoptosis and endoplasmic reticulum stress by miR-107/NCK1 axis in a FXR-dependent manner

Tauroursodeoxycholic acid (TUDCA) can activate farnesoid X receptor (FXR) to involve in the formation of gallstones. Here, this study aimed to probe the potential mechanism of TUDCA-FXR network in the formation of bile duct stone. The levels of TUDCA, FXR and NCK1 were decreased, while the level of miR-107 was increased in the serum of bile duct stone patients. FXR expression was positively correlated with TUDCA or NCK1 expression in patients, moreover, TUDCA pretreatment in biliary epithelial cells increased the levels of FXR and NCK1, and rescued the decrease of NCK1 caused by FXR knockdown in cells. Then functional analysis showed FXR knockdown caused apoptosis and endoplasmic reticulum stress (ERS) as well as suppressed proliferation in biliary epithelial cells in vitro, which were attenuated by TUDCA pretreatment or NCK1 overexpression Mechanistically, NCK1 was a target of miR-107, which was up-regulated by FXR silencing, and FXR knockdown-induced decrease of NCK1 was rescued by miR-107 inhibition. Additionally, miR-107 expression was negatively correlated with TUDCA expression in bile duct stone patients, and TUDCA pretreatment in biliary epithelial cells decreased miR-107 expression by FXR. Functionally, the pretreatment of TUDCA or FXR agonist suppressed miR-107-evoked apoptosis and ERS in biliary epithelial cells. In conclusion, TUDCA up-regulates FXR expression to activate NCK1 through absorbing miR-107, thus suppressing the apoptosis and ERS in biliary epithelial cells, these results provided a theoretical basis for elucidating the mechanism of bile duct stone formation.

FXR controls insulin content by regulating Foxa2-mediated insulin transcription

Farnesoid X receptor (FXR) is a nuclear ligand-activated receptor of bile acids that plays a role in the modulation of insulin content. However, the underlying molecular mechanisms remain unclear. Forkhead box a2 (Foxa2) is an important nuclear transcription factor in pancreatic β-cells and is involved in β-cell function. We aimed to explore the signaling mechanism downstream of FXR to regulate insulin content and underscore its association with Foxa2 and insulin gene (Ins) transcription. All experiments were conducted on FXR transgenic mice, INS-1 823/13 cells, and diabetic Goto-Kakizaki (GK) rats undergoing sham or Roux-en-Y gastric bypass (RYGB) surgery. Islets from FXR knockout mice and INS-1823/13 cells with FXR knockdown exhibited substantially lower insulin levels than that of controls. This was accompanied by decreased Foxa2 expression and Ins transcription. Conversely, FXR overexpression increased insulin content, concomitant with enhanced Foxa2 expression and Ins transcription in INS-1 823/13 cells. Moreover, FXR knockdown reduced FXR recruitment and H3K27 trimethylation in the Foxa2 promoter. Importantly, Foxa2 overexpression abrogated the adverse effects of FXR knockdown on Ins transcription and insulin content in INS-1 823/13 cells. Notably, RYGB surgery led to improved insulin content in diabetic GK rats, which was accompanied by upregulated FXR and Foxa2 expression and Ins transcription. Collectively, these data suggest that Foxa2 serves as the target gene of FXR in β-cells and mediates FXR-enhanced Ins transcription. Additionally, the upregulated FXR/Foxa2 signaling cascade could contribute to the enhanced insulin content in diabetic GK rats after RYGB.

Farnesoid X receptor represses human sulfotransferase 1A3 expression through direct binding to the promoter.

Human sulfotransferases 1A3 (SULT1A3) has received particular interest, due to their functions of catalyzing the sulfonation of numerous phenolic substrates, including bioactive endogenous molecules and therapeutic agents. However, the regulation of SULT1A3 expression and the underlying mechanism remain unclear. Here, we aimed to investigate the regulation effects of bile acid-activated farnesoid X receptor (FXR) on SULT1A3 expression, and to shed light on the mechanism thereof. Our results demonstrated that FXR agonists (CDCA and GW4064) significantly inhibit the expression of SULT1A3 at mRNA and protein levels. In addition, overexpression of FXR led to decrease in SULT1A3 expression and knockdown of FXR significantly induced the expression of SULT1A3 in protein and mRNA levels, confirming that FXR expression manifestly showed negative regulatory effect on basal SULT1A3 expression. Furthermore, a combination of luciferase reporter gene and CHIP assays showed that FXR repressed SULT1A3 transcription through direct binding to the region at base pair positions -664 to -654. In conclusion, this study for the first time confirmed FXR was a negative transcriptional regulator of human SULT1A3 enzyme.

Gamma-Muricholic Acid Inhibits Nonalcoholic Steatohepatitis: Abolishment of Steatosis-Dependent Peroxidative Impairment by FXR/SHP/LXRα/FASN Signaling.

Nonalcoholic steatohepatitis (NASH) reflects the outcome of steatosis-based peroxidative impairment. Here, the effect and mechanism of γ-muricholic acid (γ-MCA) on NASH were investigated on the basis of its actions in hepatic steatosis, lipid peroxidation, peroxidative injury, hepatocyte apoptosis, and its NAFLD activity score (NAS). The agonist action of γ-MCA on farnesoid X receptor (FXR) upregulated the small heterodimer partner (SHP) expression of hepatocytes. An increase in SHP attenuated the triglyceride-dominated hepatic steatosis which was induced in vivo by a high-fat high-cholesterol (HFHC) diet and in vitro by free fatty acids depending on the inhibition of liver X receptor α (LXRα) and fatty acid synthase (FASN). In contrast, FXR knockdown abrogated the γ-MCA-dependent lipogenic inactivation. When compared to their excessive production in HFHC diet-induced rodent NASH, products of lipid peroxidation (MDA and 4-HNE) exhibited significant reductions upon γ-MCA treatment. Moreover, the decreased levels of serum alanine aminotransferases and aspartate aminotransferases demonstrated an improvement in the peroxidative injury of hepatocytes. By TUNEL assay, injurious amelioration protected the γ-MCA-treated mice against hepatic apoptosis. The abolishment of apoptosis prevented lobular inflammation, which downregulated the incidence of NASH by lowering NAS. Collectively, γ-MCA inhibits steatosis-induced peroxidative injury to ameliorate NASH by targeting FXR/SHP/LXRα/FASN signaling.

Activation of farnesoid X receptor suppresses ER stress and inflammation via the YY1/NCK1/PERK pathway in large yellow croaker (Larimichthys crocea).

Unfolded protein responses from endoplasmic reticulum (ER) stress have been implicated in inflammatory signaling. The vicious cycle of ER stress and inflammation makes regulation even more difficult. This study examined effects of farnesoid X receptor (FXR) in ER-stress regulation in large yellow croakers. The soybean-oil-diet-induced expression of ER stress markers was decreased in fish with FXR activated. In croaker macrophages, FXR activation or overexpression significantly reduced inflammation and ER stress caused by tunicamycin (TM), which was exacerbated by FXR knockdown. Further investigation showed that the TM-induced phosphorylation of PERK and EIF2α was inhibited by the overexpression of croaker FXR, and it was increased by FXR knockdown. Croaker NCK1 was then confirmed to be a regulator of PERK, and its expression in macrophages is increased by FXR overexpression and decreased by FXR knockdown. The promoter activity of croaker NCK1 was inhibited by yin-yang 1 (YY1). Furthermore, the results show that croaker FXR overexpression could suppress the P65-induced promoter activity of YY1 in HEK293t cells and decrease the TM-induced expression of yy1 in macrophages. These results indicate that FXR could suppress P65-induced yy1 expression and then increase NCK1 expression, thereby inhibiting the PERK pathway. This study may benefit the understanding of ER stress regulation in fish, demonstrating that FXR can be used in large yellow croakers as an effective target for regulating ER stress and inflammation.

Diminished Tubule Epithelial Farnesoid X Receptor Expression Exacerbates Inflammation and Fibrosis Response in Aged Rat Kidney.

The age-associated functional decline of the kidney is accompanied by structural changes including glomerular sclerosis and interstitial fibrosis. Aging kidneys also exhibit increased vulnerability in stressful environmental conditions. In this study, we assessed the differences in responses between young and aged animals to folic acid (FA)-induced renal fibrosis. To monitor the effects of aging on FA-induced kidney fibrosis, we administered FA (250 mg/kg) to young (6-month old) and aged (20-month old) rats. The development of severe fibrosis was only detected in aged rat kidneys, which was accompanied by increased kidney injury and inflammation. Furthermore, we found that FA-treated aged rats had significantly lower farnesoid X receptor (FXR) expression in the tubular epithelial cells than the rats not treated with FA. Interestingly, the extent of inflammation was severe in the kidneys of aged rat, where the FXR expression was low. To explore the role of FXR in kidney inflammation, in vitro studies were performed using NRK52E kidney tubule epithelial cells. NF-κB activation by lipopolysaccharide treatment induces chemokine production in NRK52E cells. The activation of FXR by obeticholic acid significantly reduced the transcriptional activity of NF-κB and chemokine production. In contrast, FXR knockdown increased LPS-induced chemokine production in NRK52E cells. Finally, FXR-knockout mice that were administered FA showed increased inflammation and severe fibrosis. In summary, we demonstrated that diminished FXR expression in the epithelial cells of the renal tubules exacerbated the fibrotic response in aged rat kidneys by upregulating pro-inflammatory NF-κB activation.

Mebhydrolin ameliorates glucose homeostasis in type 2 diabetic mice by functioning as a selective FXR antagonist

IntroductionType 2 diabetes mellitus (T2DM) is a chronic disease with hallmarks of hyperglycemia and hyperlipidemia. Long-term hyperglycemia damages the functions of multiple tissues and organs leading to a series of complications and disability or even death. Nuclear receptor farnesoid X receptor (FXR) antagonism has been recently discovered to exhibit beneficial effect on glucose metabolism in T2DM mice, although the underlying mechanisms remain unclear. Here, we performed the study on the discovery of new FXR antagonist and investigated the mechanism underlying the amelioration of FXR antagonism on glucose homeostasis in T2DM mice by using the determined FXR antagonist as a probe. MethodsFXR antagonist Mebhydrolin was discovered by screening against the lab in-house FDA approved drug library through surface plasmon resonance (SPR), microscale thermophoresis (MST), AlphaScreen, mammalian one-hybrid and transactivation assays. Activity of Mebhydrolin in improving glucose homeostasis was evaluated in db/db and HFD/STZ-induced T2DM mice, and the mechanisms governing the regulation of Mebhydrolin were investigated by assays of immunostaining, Western blot, ELISA, RT-PCR against liver tissues of both T2DM mice and the T2DM mice with liver-specific FXR knockdown injected via adeno-associated-virus AAV-FXR-RNAi and mouse primary hepatocytes. Finally, molecular docking and molecular dynamics (MD) technology-based study was performed to investigate the structural basis for the antagonistic regulation of Mebhydrolin against FXR at an atomic level. FindingsMebhydrolin ameliorated blood glucose homeostasis in T2DM mice by both suppressing hepatic gluconeogenesis via FXR/miR-22-3p/PI3K/AKT/FoxO1 pathway and promoting glycogen synthesis through FXR/miR-22-3p/PI3K/AKT/GSK3β pathway. Structurally, residues L291, M332 and Y373 of FXR were required for Mebhydrolin binding to FXR-LBD, and Mebhydrolin induced H2 and H6 shifting of FXR potently affecting the regulation of the downstream target genes. ConclusionsOur work has revealed a novel mode for the regulation of FXR against glucose metabolism in T2DM mice and highlighted the potential of Mebhydrolin in the treatment of T2DM.

Activation of farnesoid X receptor (FXR) induces crystallin zeta expression in mouse medullary collecting duct cells

Crystallin zeta (CRYZ) is a phylogenetically restricted water-soluble protein and provides cytoprotection against oxidative stress via multiple mechanisms. Increasing evidence suggests that CRYZ is high abundantly expressed in the kidney where it acts as a transacting factor in increasing glutaminolysis and the Na+/K+/2Cl- cotransporter (BSC1/NKCC2) expression to help maintain acid-base balance and medullary hyperosmotic gradient. However, the mechanism by which CRYZ is regulated in the kidney remains largely uncharacterized. Here, we show that CRYZ is a direct target of farnesoid X receptor (FXR), a nuclear receptor important for renal physiology. We found that CRYZ was ubiquitously expressed in mouse kidney and constitutively expressed in the cytoplasm of medullary collecting duct cells (MCDs). In primary cultured mouse MCDs, CRYZ expression was significantly upregulated by the activation and overexpression of FXR. FXR-induced CRYZ expression was almost completely abolished in the MCD cells with siRNA-mediated FXR knockdown. Consistently, treatment with FXR agonists failed to induce CRYZ expression in the MCDs isolated from mice with global and collecting duct-specific FXR deficiency. We identified a putative FXR response element (FXRE) on the CRYZ gene promoter. The luciferase reporter and ChIP assays revealed that FXR can bind directly to the FXRE site, which was further markedly enhanced by FXR activation. Furthermore, we found CRYZ overexpression in MCDs significantly attenuated hypertonicity-induced cell death possibly via increasing Bcl-2 expression. Collectively, our findings demonstrate that CRYZ is constitutively expressed in renal medullary collecting duct cells, where it is transcriptionally controlled by FXR. Given a critical role of FXR in MCDs, CRYZ may be responsible for protective effect of FXR on the survival of MCDs under hypertonic condition during dehydration.

Activation of the Farnesoid X Receptor (FXR) Suppresses Linoleic Acid-Induced Inflammation in the Large Yellow Croaker (Larimichthys crocea)

High linoleic acid (LA) intake leads to inflammation that adversely influences health in fish. However, whether the farnesoid X receptor (FXR) could be an effective target for regulating LA-induced inflammation remains unknown. The purpose of this study was to investigate the role of FXR in the regulation of LA-induced inflammation in large yellow croakers. Large yellow croakers (initial weight of 10.03±0.02 g) were allocated to 4 groups and fed a fish oil diet (6% FO), a soybean oil diet (6% SO), or the SO diet supplemented with 300 or 900 mg chenodeoxycholic acid (CDCA)/kg for 10 wk. The cultured kidney cell line PCK and primary hepatocytes from large yellow croakers were stimulated by LA (50 μM) after pretreatment with an FXR ligand (GW4064 or CDCA) or transfection with fxr-small interfering RNA (siFXR). mRNA expression of proinflammatory genes in the head kidney and liver tissues, PCK cells, and primary hepatocytes was determined by qPCR. The luciferase reporter assay, electrophoretic mobility shift assay, and immunoprecipitation assay were conducted in HEK 293T cells to determine the transcriptional activity of P65 and protein interactions between P65 and FXR or the small heterodimer partner (SHP). Proinflammatory genes were 93-1180% higher in the SO group compared with the FO group. CDCA supplementation decreased mRNA expression of proinflammatory genes by 17-87% while increasing fxr and shp expression by 120-460%. In PCK cells and primary hepatocytes, ligand-mediated activation of FXR decreased the LA-induced expression of proinflammatory genes by 18-67%, whereas siRNA-mediated knockdown of FXR increased the LA-induced expression of proinflammatory genes by 64-96%. FXR bound to the promoter of shp and regulated its mRNA expression. Both FXR and SHP could bind to P65 to suppress the transcriptional activity of P65. These results indicate that FXR has anti-inflammatory properties in large yellow croakers by directly and indirectly suppressing NFκB activity.

Diabetes downregulates peptide transporter 1 in the rat jejunum: possible involvement of cholate-induced FXR activation.

Peptide transporter 1 (PepT1), highly expressed on the apical membrane of enterocytes, is involved in energy balance and mediates intestinal absorption of peptidomimetic drugs. In this study, we investigated whether and how diabetes affected the function and expression of intestinal PepT1. Diabetes was induced in rats by combination of high-fat diet and low dose streptozocin injection. Pharmacokinetics study demonstrated that diabetes significantly decreased plasma exposures of cephalexin and acyclovir following oral administration of cephalexin and valacyclovir, respectively. Single-pass intestinal perfusion analysis showed that diabetes remarkably decreased cephalexin absorption, which was associated with decreased expression of intestinal PepT1 protein. We assessed the levels of bile acids in intestine of diabetic rats, and found that diabetic rats exhibited significantly higher levels of chenodeoxycholic acid (CDCA), cholic acid (CA) and glycocholic acid (GCA), and lower levels of lithocholic acid (LCA) and hyodeoxycholic acid (HDCA) than control rats; intestinal deoxycholic acid (DCA) levels were unaltered. In Caco-2 cells, the 6 bile acids remarkably decreased expression of PepT1 protein with CDCA causing the strongest inhibition, whereas TNF-α, LPS and insulin little affected expression of PepT1 protein; short-chain fatty acids induced rather than decreased expression of PepT1 protein. Farnesoid X receptor (FXR) inhibitor glycine-β-muricholic acid or FXR knockdown reversed the downregulation of PepT1 expression by CDCA and GW4064 (another FXR agonist). In diabetic rats, the expression of intestinal FXR protein was markedly increased. Oral administration of CDCA (90, 180 mg·kg-1·d-1, for 3 weeks) dose-dependently decreased the expression and function of intestinal PepT1 in rats. In conclusion, diabetes impairs the expression and function of intestinal PepT1 partly via CDCA-mediated FXR activation.

Ursodeoxycholic Acid (UDCA) Promotes Lactate Metabolism in Mouse Hepatocytes through Cholic Acid (CA) - Farnesoid X Receptor (FXR) Pathway.

Persistent hyperlactatemia is associated with greater mortality in shock. Liver is the main site of lactate metabolism. In the first part, freshly isolated hepatocytes were incubated in 10% fetal bovine serum William's E medium supplemented with 10 mM lactate. Cells were then exposed to 100 μM ursodeoxycholic acid (UDCA), with no addition (control) for 2, 4, 6, 8 h. In the second part, hepatocytes were treated with Silencer select siRNA targeting FXR or scramble siRNA. The siRNA treatment was repeated twenty four hours later, and the cells were used in the experiments twenty-four hours after the second treatment. Then hepatocytes were incubated in 10% fetal bovine serum William's E medium supplemented with 10 mM lactate. Cells were then exposed to 100 μM UDCA for 2, 4, 6, 8 h. Lactate concentration was determined by ABL80 automatic blood gas analyzer. UDCA increased ability of hepatocytes to remove lactate. After the knockdown of FXR, effects caused by UDCA were weakened. These results demonstrate that UDCA promotes lactate metabolism in mouse hepatocytes through CA-FXR pathway.

Farnesoid X receptor activation induces the degradation of hepatotoxic 1-deoxysphingolipids in non-alcoholic fatty liver disease.

Patients with non-alcoholic fatty liver disease (NAFLD) exhibit higher levels of plasma 1-deoxysphingolipids than healthy individuals. The aim of this study was to investigate the role of farnesoid X receptor (FXR) in 1-deoxysphingolipid de novo synthesis and degradation. Mice were fed with a high-fat diet (HFD) to induce obesity and NAFLD, and then treated with the FXR ligand obeticholic acid (OCA). Histology and gene expression analysis were performed on liver tissue. Sphingolipid patterns from NAFLD patients and mouse models were assessed by liquid chromatography-mass spectrometry. The molecular mechanism underlying the effect of FXR activation on sphingolipid metabolism was studied in Huh7 cells and primary cultured hepatocytes, as well as in a 1-deoxysphinganine-treated mouse model. 1-deoxysphingolipids were increased in both NAFLD patients and mouse models. FXR activation by OCA protected the liver against oxidative stress, apoptosis, and reduced 1-deoxysphingolipid levels, both in a HFD-induced mouse model of obesity and in 1-deoxysphinganine-treated mice. In vitro, FXR activation lowered intracellular 1-deoxysphingolipid levels by inducing Cyp4f-mediated degradation, but not by inhibiting de novo synthesis, thereby protecting hepatocytes against doxSA-induced cytotoxicity, mitochondrial damage, and apoptosis. Overexpression of Cyp4f13 in cells was sufficient to ameliorate doxSA-induced cytotoxicity. Treatment with the Cyp4f pan-inhibitor HET0016 or FXR knock-down fully abolished the protective effect of OCA, indicating that OCA-mediated 1-deoxysphingolipid degradation is FXR and Cyp4f dependent. Our study identifies FXR-Cyp4f as a novel regulatory pathway for 1-deoxysphingolipid metabolism. FXR activation represents a promising therapeutic strategy for patients with metabolic syndrome and NAFLD.

FXR Mediates Adenylyl Cyclase 8 Expression in Pancreatic β-Cells.

Adenylyl cyclase 8 (ADCY8) and Farnesoid X Receptor (FXR) have been identified in pancreatic β-cells and play important roles in insulin secretion. But the mechanisms underlying with respect to the regulation of ADCY8 expression in β-cells, particularly whether FXR is involved, remain unexplored. We now show that ADCY8 expression is decreased in Goto-Kakizaki (GK) rat islets compared with healthy Wistar controls. We also found that reduced ADCY8 is associated with decreased expression of FXR. Consistently, ADCY8 expression was suppressed by the knockdown of FXR in INS-1 832/13 cells, as well as the islets from FXR knockout mice. On the contrary, ADCY8 expression was increased in FXR-overexpressed INS-1 832/13 cells or in the case of FXR activation. Mechanistically, FXR directly binds to Adcy8 promoter and recruits the histone acetyltransferase Steroid Receptor Coactivator 1 (SRC1), thereby resulting in the increased acetylation of histone H3 in Adcy8 locus, promoting Adcy8 gene transcription in β-cells. Thus, this study indicates that FXR is a critical transcription factor that mediates ADCY8 expression in pancreatic β-cells and has characterized the chromatin modification associated with Adcy8 transcription.

Bile acids promote gastric intestinal metaplasia by upregulating CDX2 and MUC2 expression via the FXR/NF-κB signalling pathway

Bile acids serve a critical role in the induction of gastric intestinal metaplasia (IM) and gastric carcinogenesis. The present study investigated the effects of bile acids on the induction of gastric IM formation. The results demonstrated that the expression levels of caudal-related homeobox transcription factor 2 (CDX2), mucin 2 (MUC2) and farnesoid X receptor (FXR) were increased in vitro and in vivo following treatment with bile acids, and CDX2 transcriptionally activated MUC2 expression. Furthermore, knockdown of FXR attenuated bile acid-enhanced CDX2 promoter activity and protein expression. Conversely, the FXR agonist GW4064 synergistically enhanced bile acid-induced CDX2 promoter activity. Bile acid treatment led to an increase in nuclear factor (NF)-κB activity and protein expression. Treatment with GW4064 or the FXR antagonist Z-guggulsterone enhanced or attenuated bile acid-induced NF-κB activity, respectively. In addition, quantitative chromatin immunoprecipitation confirmed that bile acids led to enhanced binding of p50 to the CDX2 promoter, whereas this effect was not observed for p65. Treatment with GW4064 or Z-guggulsterone enhanced and attenuated the binding activity of p50 to the CDX2 promoter, respectively. These results indicated that bile acids may activate the FXR/NF-κB signalling pathway, thereby upregulating CDX2 and MUC2 expression in normal gastric epithelial cells.

Farnesoid X receptor signaling activates the hepatic X-box binding protein 1 pathway in vitro and in mice.

FXR signaling activates the IRE1α/XBP1 pathway in vivo and in vitro. FXR pathway activation increases XBP1 splicing and enhances p-IRE1α expression. These effects are mediated, at least in part, by SHP. IRE1α/XBP1 pathway activation by bile acids and pharmacological FXR agonists may be protective during liver injury and may have therapeutic implications for liver diseases. (Hepatology 2018;68:304-316).

AMPK Phosphorylation of FXR at Threonine Controls Hepatic Glucose Production

Farnesoid X receptor (FXR) controls the expression of genes involved in the regulation of energy metabolism. Nonetheless, the regulatory signal of FXR in glucose‐lowering effect and the underlying mechanisms are largely unknown. Based on the functional link between SIRT1 and AMPK, this study explored the effect of AMPK on the phosphorylation of FXR and its role for SIRT1‐mediated hepatic glucose metabolism. SIRT1 overexpression increased the transcriptional activities of FXR response reporter gene, whereas a loss of SIRT1 had the opposite effect. SIRT1‐mediated FXR transactivation was significantly blocked by the inhibition of AMPK. The impact of SIRT1 and AMPK on the regulation of FXR function was strengthened by the pharmacological effect of ajoene. We also found that AMPK directly phosphorylated FXR at Thr156 residue as shown by in vitro kinase, oligopeptide competition and point‐mutation analyses. The overexpression of FXR‐T156A mutant attenuated AMPK‐mediated FXR phosphorylation and the binding of FXR to coactivator. The inhibition of glucose‐6‐phosphatase expression or hepatocyte glucose production by CA‐AMPK or ajoene was abrogated by either FXR knockdown or FXR‐T156A mutant overexpression. Finally, ajoene treatment diminished hyperglycemia and sensitized insulin signaling in high fat diet‐induced obese mice. In conclusion, AMPK phosphorylates FXR at Thr156 downstream of SIRT1, which may contribute to the inhibition of hepatic glucose production.Support or Funding InformationThis research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (NRF‐2015R1A2A1A10052663) and the Basic Science Research Program through NRF funded by the Ministry of Education (2017R1D1A1B03028272).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of FXR promotes intestinal metaplasia of gastric cells via SHP‑dependent upregulation of the expression of CDX2

Gastric intestinal metaplasia (IM) induced by bile acid is a precancerous lesion of gastric adenocarcinoma and is associated with the expression of caudal-related homeobox 2 (CDX2). In the present study, the role of farnesoid X receptor (FXR) on the regulation of CDX2 in gastric cells was investigated and the underlying molecular mechanisms were examined. Human gastric cell lines were treated with chenodeoxycholic acid (CDCA) or FXR agonist GW4064. Cells were treated with CDCA in the presence or absence of the FXR antagonist or FXR siRNA transfection. Next, cells were treated with CDCA in the presence or absence of SHP siRNA transfection and FXR, CDX2 and SHP mRNA and protein levels were determined by reverse transcription-quantitative polymerase chain reaction and western blot analysis. A chromatin immunoprecipitation assay was performed to examine the relationship between FXR and SHP and the expressions of FXR and CDX2 in gastritis and IM tissues were detected using immunohistochemistry. The results revealed that CDCA was able to induce CDX2 expression, which could be blocked by inhibition or knockdown of FXR. Mechanistically, FXR directly induced the expression of small heterodimer partner (SHP). SHP knockdown significantly decreased CDCA-induced CDX2 expression. ChIP results indicated that FXR could directly bind SHP promoter and promote SHP expression. Finally, immunohistochemistry results demonstrated that the expression levels of CDX2 and FXR in human IM lesions were significantly higher, compared with those in gastritis lesions, and were positively correlated. Collectively, these results revealed that the activation of FXR and sequential direct transcriptional induction of SHP were involved in the expression of CDX2 induced by bile acid in gastric IM lesions.

Farnesoid X receptor regulates SULT1E1 expression through inhibition of PGC1α binding to HNF4α

Sulfotransferase 1E1 (SULT1E1, also known as estrogen sulfotransferase) plays an important role in metabolism and detoxification of many endogenous and exogenous compounds (e.g., estrogens and flavonoids). Here we aimed to assess the effects of farnesoid X receptor (FXR) activation on SULT1E1 expression, and to determine the mechanism thereof. Treatment with specific FXR agonists (i.e., GW4064 and CDCA) significantly decreased both mRNA and protein levels of SULT1E1 in HepG2 cells. This was accompanied by a decrease in the enzymatic activity. The inhibitory effect was potentiated by FXR overexpression but attenuated by FXR knockdown, confirming FXR-dependent regulation of SULT1E1. Surprisingly, direct regulation of SULT1E1 by FXR was unlikely because FXR did not bind to SULT1E1 promoter or enhancer as revealed by chromatin immunoprecipitation (ChIP). Interestingly, SULT1E1 regulation was abolished when HNF4α (hepatocyte nuclear factor 4α, a known activator of SULT1E1) was silenced, supporting a critical role for HNF4α in FXR regulation of SULT1E1. Furthermore, a combination of ChIP, luciferase reporter and co-immunoprecipitation assays showed that FXR inhibited HNF4α transactivation of SULT1E1 by suppressed binding of the co-activator PGC1α (peroxisome proliferator-activated receptor-γ coactivator 1α) to HNF4α. In conclusion, FXR transcriptionally regulates SULT1E1 through inhibition of PGC1α binding to HNF4α. Targeting the FXR-SULT1E1 axis may represent a promising approach for management of estrogen-related diseases.

Farnesoid X receptor, a novel proto-oncogene in non-small cell lung cancer, promotes tumor growth via directly transactivating CCND1

Farnesoid X receptor (FXR), a nuclear receptor for maintaining bile acid homeostasis, has been recognized as a tumor suppressor in enterohepatic tissues. However, its expression and functional role in non-small cell lung cancer (NSCLC) remain unclear. We report that FXR is significantly increased in NSCLC and that it predicts poor clinical outcomes in NSCLC patients. FXR knockdown in NSCLC cells inhibited in vitro cell proliferation, blocked xenograft growth in nude mice, and delayed the G1/S transition of the cell cycle, whereas ectopic overexpression of FXR promoted NSCLC cell proliferation. Mechanistic analysis demonstrated that FXR could directly bind to an inverted repeat-0 sequence in the CCND1 promoter and activate its transcription. Cyclin D1 overexpression rescued NSCLC cells from the delayed G1/S transition and the impaired cell proliferation induced by FXR knockdown. Importantly, a positive correlation between the expression of FXR and cyclin D1 was confirmed in NSCLC samples, and patients with high expression of both FXR and cyclin D1 had the worst prognosis. In summary, our results suggest that FXR has oncogenic potential in NSCLC development, providing mechanistic insights that could be exploited for both prognostic and therapeutic purposes.

Transcriptional Regulation of Human UDP-Glucuronosyltransferase 2B10 by Farnesoid X Receptor in Human Hepatoma HepG2 Cells.

Little is known about transcriptional regulators of UDP-glucuronosyltransferase 2B10 (UGT2B10), an enzyme known to glucuronidate many chemicals and drugs such as nicotine and tricyclic antidepressants. Here, we uncovered that UGT2B10 was transcriptionally regulated by farnesoid X receptor (FXR), the bile acid sensing nuclear receptor. GW4064 and chenodeoxycholic acid (two specific FXR agonists) treatment of HepG2 cells led to a significant increase in the mRNA level of UGT2B10. The treated cells also showed enhanced glucuronidation activities toward amitriptyline (an UGT2B10 probe substrate). In reporter gene assays, the extent of UGT2B10 activation by the FXR agonists was positively correlated with the amount of cotransfected FXR. Consistently, knockdown of FXR by shRNA attenuated the induction effect on UGT2B10 expression. Furthermore, a combination of electrophoretic mobility shift assay and chromatin immunoprecipitation showed that the FXR receptor trans-activated UGT2B10 through its specific binding to the -209- to -197-bp region (an IR1 element) of the UGT2B10 promoter. In summary, our results for the first time established FXR as a transcriptional regulator of human UGT2B10.