Ann M. Thomas

Genome-Wide Tissue-Specific Farnesoid X Receptor Binding in Mouse Liver and Intestine

Farnesoid X receptor (FXR) is a bile acid‐activated transcription factor belonging to the nuclear receptor superfamily. FXR is highly expressed in liver and intestine and crosstalk mediated by FXR in these two organs is critical in maintaining bile acid homeostasis. FXR deficiency has been implicated in many liver and intestine diseases. However, regulation of transcription by FXR at the genomic level is not known. This study analyzed genome‐wide FXR binding in liver and intestine of mice treated with a synthetic FXR ligand (GW4064) by chromatin immunoprecipitation coupled to massively parallel sequencing (ChIP‐seq). The results showed a large degree of tissue‐specific FXR binding, with only 11% of total sites shared between liver and intestine. The sites were widely distributed between intergenic, upstream, intragenic, and downstream of genes, with novel sites identified within even known FXR target genes. Motif analysis revealed a half nuclear receptor binding site, normally bound by a few orphan nuclear receptors, adjacent to the FXR response elements, indicating possible involvement of some orphan nuclear receptors in modulating FXR function. Furthermore, pathway analysis indicated that FXR may be extensively involved in multiple cellular metabolic pathways. Conclusion: This study reports genome‐wide FXR binding in vivo and the results clearly demonstrate tissue‐specific FXR/gene interaction. In addition, FXR may be involved in regulating broader biological pathways in maintaining hepatic and intestinal homeostasis. (HEPATOLOGY 2010.)

Farnesoid X Receptor Deficiency in Mice Leads to Increased Intestinal Epithelial Cell Proliferation and Tumor Development

Increased dietary fat consumption is associated with colon cancer development. The exact mechanism by which fat induces colon cancer is not clear, however, increased bile acid excretion in response to high-fat diet may promote colon carcinogenesis. The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily, and bile acids are endogenous ligands of FXR. FXR is highly expressed in the intestine and liver where FXR is essential for maintaining bile acid homeostasis. The role of FXR in intestine cancer development is not known. The current study evaluated the effects of FXR deficiency in mice on intestinal cell proliferation and cancer development. The results showed that FXR deficiency resulted in increased colon cell proliferation, which was accompanied by an up-regulation in the expression of genes involved in cell cycle progression and inflammation, including cyclin D1 and interleukin-6. Most importantly, FXR deficiency led to an increase in the size of small intestine adenocarcinomas in adenomatous polyposis coli mutant mice. Furthermore, after treatment with a colon carcinogen, azoxymethane, FXR deficiency increased the adenocarcinoma multiplicity and size in colon and rectum of C57BL/6 mice. Loss of FXR function also increased the intestinal lymphoid nodule numbers in the intestine. Taken together, the current study is the first to show that FXR deficiency promotes cell proliferation, inflammation, and tumorigenesis in the intestine, suggesting that activation of FXR by nonbile acid ligands may protect against intestinal carcinogenesis.

Increased Activation of the Wnt/β-Catenin Pathway in Spontaneous Hepatocellular Carcinoma Observed in Farnesoid X Receptor Knockout Mice

Farnesoid X receptor (FXR), the primary bile acid-sensing nuclear receptor, also is known for its anticancer properties. It is known that FXR deficiency in mice results in spontaneous hepatocellular carcinoma (HCC), but the mechanisms are not completely understood. We report that sustained activation of the Wnt/β-catenin pathway is associated with spontaneous HCC in FXR-knockout (KO) mice. HCC development was studied in FXR-KO mice at 3, 8, and 14 months of age. No tumors were observed at either 3 or 8 months, but the presence of HCC was observed in 100% of the FXR-KO mice at the age of 14 months. Further analysis revealed no change in β-catenin activation in the livers of 3-month-old FXR-KO mice, but a moderate increase was observed in 8-month-old FXR-KO mice. β-Catenin activation further increased significantly in 14-month-old tumor-bearing mice. Further analysis revealed that two independent mechanisms might be involved in β-catenin activation in the livers of FXR-KO mice. Activation of canonical Wnt signaling was evident as indicated by increased Wnt4 and dishevelled expression along with glycogen synthase kinase-3β inactivation. We also observed decreased expression of E-cadherin, a known regulator of β-catenin, in FXR-KO mice. The decrease in E-cadherin expression was accompanied by increased expression of its transcriptional repressor, Snail. Consistent with the increased HCC in FXR-KO mice, we observed a significant decrease in FXR expression and activity in human HCC samples. Taken together, these data indicate that a temporal increase in the activation of Wnt/β-catenin is observed during spontaneous HCC development in FXR-KO mice and is potentially critical for tumor development.

Farnesoid X receptor induces murine scavenger receptor Class B type I via intron binding.

Farnesoid X receptor (FXR) is a nuclear receptor and a key regulator of liver cholesterol and triglyceride homeostasis. Scavenger receptor class B type I (SR-BI) is critical for reverse cholesterol transport (RCT) by transporting high-density lipoprotein (HDL) into liver. FXR induces SR-BI, however, the underlying molecular mechanism of this induction is not known. The current study confirmed induction of SR-BI mRNA by activated FXR in mouse livers, a human hepatoma cell line, and primary human hepatocytes. Genome-wide FXR binding analysis in mouse livers identified 4 putative FXR response elements in the form of inverse repeat separated by one nucleotide (IR1) at the first intron and 1 IR1 at the downstream of the mouse Sr-bi gene. ChIP-qPCR analysis revealed FXR binding to only the intronic IR1s, but not the downstream one. Luciferase assays and site-directed mutagenesis further showed that 3 out of 4 IR1s were able to activate gene transcription. A 16-week high-fat diet (HFD) feeding in mice increased hepatic Sr-bi gene expression in a FXR-dependent manner. In addition, FXR bound to the 3 bona fide IR1s in vivo, which was increased following HFD feeding. Serum total and HDL cholesterol levels were increased in FXR knockout mice fed the HFD, compared to wild-type mice. In conclusion, the Sr-bi/SR-BI gene is confirmed as a FXR target gene in both mice and humans, and at least in mice, induction of Sr-bi by FXR is via binding to intronic IR1s. This study suggests that FXR may serve as a promising molecular target for increasing reverse cholesterol transport.

Role of Nuclear Receptors in Lipid Dysfunction and Obesity-Related Diseases

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 12 meeting in San Diego, CA. The presentations discussed the roles of a number of nuclear receptors in regulating glucose and lipid homeostasis, the pathophysiology of obesity-related disease states, and the promise associated with targeting their activities to treat these diseases. While many of these receptors (in particular, constitutive androstane receptor and pregnane X receptor) and their target enzymes have been thought of as regulators of drug and xenobiotic metabolism, this symposium highlighted the advances made in our understanding of the endogenous functions of these receptors. Similarly, as we gain a better understanding of the mechanisms underlying bile acid signaling pathways in the regulation of body weight and glucose homeostasis, we see the importance of using complementary approaches to elucidate this fascinating network of pathways. The observation that some receptors, like the farnesoid X receptor, can function in a tissue-specific manner via well defined mechanisms has important clinical implications, particularly in the treatment of liver diseases. Finally, the novel findings that agents that selectively activate estrogen receptor β can effectively inhibit weight gain in a high-fat diet model of obesity identifies a new role for this member of the steroid superfamily. Taken together, the significant findings reported during this symposium illustrate the promise associated with targeting a number of nuclear receptors for the development of new therapies to treat obesity and other metabolic disorders.

Alanine-glutamine dipeptide (AGD) inhibits expression of inflammation-related genes in hemorrhagic shock

BACKGROUND Inflammatory factors play an important role in the production of cellular damage after shock and reperfusion. Glutamine has been used to modulate the inflammatory response. Alanine-glutamine dipeptide (AGD) is a glutamine source. The hypothesis of the present study is that AGD given during resuscitation will suppress postshock expression of messenger ribonucleic acid (mRNA) for tumor necrosis factor (TNF-alpha), interleukin-1 (IL-1beta) and inducible nitric oxide synthase (iNOS). METHODS Male Sprague-Dawley rats (n = 74, 350 g +/- 30 g) were randomly assigned to 5 groups. Under isoflurane anesthesia, the femoral artery and vein were cannulated. Hemorrhagic shock was induced by withdrawing blood through the arterial cannula until the mean arterial pressure (MAP) was 25-30 mm Hg and maintained at the level for 30 minutes with further withdrawals. Resuscitation was carried out by giving 21 mL/kg Ringers lactate (LR) with or without the administration of AGD (936 mg/kg) and returning the shed blood. Controls were normal (anesthesia only), sham (surgical preparation), and shock (preparation and shock). Rats (n = 45, 9 per group) were killed 30 minutes after completion of resuscitation. Liver samples were collected, and total RNA was isolated for reverse transcription-polymerase chain reaction analysis of mRNA (TNF-alpha, IL-1beta, iNOS, and beta-actin). RESULTS MAP recovered more quickly in the AGD group than in the LR group. Increased expression of liver mRNA for TNF-alpha, IL-1beta, and iNOS was seen after hemorrhagic shock and resuscitation. AGD treatment significantly reduced mRNA expression for all 3. CONCLUSIONS AGD modified the expression of genes controlling cytokines and iNOS in the liver. This agent is a potential treatment for hemorrhagic shock.

Tissue Specific Induction of p62/Sqstm1 by Farnesoid X Receptor

Background Farnesoid X Receptor (FXR) is a member of the nuclear receptor superfamily and is a ligand-activated transcription factor essential for maintaining liver and intestinal homeostasis. FXR is protective against carcinogenesis and inflammation in liver and intestine as demonstrated by the development of inflammation and tumors in the liver and intestine of FXR knock-out mice. However, mechanisms for the protective effects of FXR are not completely understood. This study reports a novel role of FXR in regulating expression of Sqstm1, which encodes for p62 protein. p62 plays an important role in maintaining cellular homeostasis through selective autophagy and activating signal transduction pathways, such as NF-κB to support cell survival and caspase-8 to initiate apoptosis. FXR regulation of Sqstm1 may serve as a protective mechanism. Methods and Results This study showed that FXR bound to the Sqstm1 gene in both mouse livers and ileums as determined by chromatin immunoprecipitation. In addition, FXR activation enhanced transcriptional activation of Sqstm1 in vitro. However, wild-type mice treated with GW4064, a synthetic FXR ligand, showed that FXR activation induced mRNA and protein expression of Sqstm1/p62 in ileum, but not in liver. Interestingly, FXR-transgenic mice showed induced mRNA expression of Sqstm1 in both liver and ileum compared to wild-type mice. Conclusions Our current study has identified a novel role of FXR in regulating the expression of p62, a key factor in protein degradation and cell signaling. Regulation of p62 by FXR indicates tissue-specific and gene-dosage effects. Furthermore, FXR-mediated induction of p62 may implicate a protective mechanism of FXR.

Crocetin Inhibits mRNA Expression for Tumor Necrosis Factor-α, Interleukin-1β, and Inducible Nitric Oxide Synthase in Hemorrhagic Shock

BACKGROUND Inflammatory factors play an important role in cellular damage after shock and resuscitation. Crocetin, a saffron-derived carotenoid, has been shown to improve postshock recovery of cellular adenosine triphosphate (ATP) and to increase overall survival in an experimental model of hemorrhagic shock. The hypothesis of the present study is that treatment with crocetin at the beginning of resuscitation suppresses subsequent expression of messenger ribonucleic acid (mRNA) for tumor necrosis factor (TNF-α), interleukin-1 (IL-1β) and inducible nitric oxide synthase (iNOS). METHODS Male Sprague-Dawley rats (n = 45, 350 ± 30 g) were randomly assigned to 5 groups of 9 animals each. After anesthesia with isoflurane, the femoral artery and vein were surgically cannulated. Hemorrhagic shock was induced by withdrawing blood through the arterial cannula until the mean arterial pressure (MAP) was 25-30 mm Hg and maintained at the level for 30 minutes with further withdrawals. Resuscitation was carried out by giving 21 mL/kg Ringers lactate (LR) and returning the shed blood, with or without the initial administration of crocetin (2 mg/kg). Controls were normal (anesthesia only), sham (surgical preparation), and shock (preparation and shock). Rats were killed 30 minutes after completion of resuscitation. Liver samples were collected for reverse transcription-polymerase chain reaction (RT-PCR) analysis of mRNA (TNF-α, IL-1β, iNOS, and β-actin). RESULTS Liver mRNA expression for TNF-α, IL-1β, and iNOS was found in more animals in the shock and shock-plus-resuscitation groups than in the sham control group. The group resuscitated from shock with crocetin had mRNA expression for TNF-α, IL-1β, and iNOS in fewer animals than either of the other shock groups and was no different from the sham control group. CONCLUSIONS Crocetin modified the hepatic mRNA expression of cytokines and iNOS in a shock model. This agent continues to show promise as a potential treatment for hemorrhagic shock.

Hepatocyte Nuclear Factor 4 Alpha and Farnesoid X Receptor Co-regulates Gene Transcription in Mouse Livers on a Genome-wide Scale

PurposeFarnesoid X receptor (Fxr) is a ligand-activated nuclear receptor critical for liver function. Reports indicate that the functions of Fxr in the liver may overlap with those of hepatocyte nuclear factor 4α (Hnf4α), but studies of their precise genome-wide interaction to regulate gene transcription in the liver are lacking. Thus, we compared the genome-wide binding of Fxr and Hnf4α in the liver of mice and characterized their cooperative activity on binding to and activating target gene transcription.MethodsGenome-wide ChIP-Seq data of Fxr and Hnf4α in mouse liver were analyzed by MACS, BEDTools, and DAVID. Co-immunoprecipitation, ChIP-qPCR, and luciferase assays were done to test for protein-protein interaction and cooperative binding.ResultsChIP-seq analysis showed nearly 50% binding sites of Fxr and Hnf4α in mouse liver overlap and Hnf4α bound to shared target sites upstream and in close proximity to Fxr. Moreover, genes co-bound by Fxr and Hnf4α are enriched in complement and coagulation cascades and drug metabolism. A direct Fxr-Hnf4α protein interaction dependent on Fxr activity was detected and transcriptional assays suggest that Hnf4α can increase Fxr transcriptional activity. Conversely, binding assays showed Hnf4α can be either Fxr-dependent or -independent at different shared binding sites.ConclusionOur results showed that Fxr cooperates with Hnf4α in the liver to modulate gene transcription. This study provides the first evidence on a genome-wide scale of both cooperative and independent interactions between Fxr and Hnf4α in regulating gene transcription in the liver.

Crocetin reduces activation of hepatic apoptotic pathways and improves survival in experimental hemorrhagic shock.

BACKGROUND Hemorrhagic shock results in cellular damage and cell death. A primary mechanism is cellular apoptosis from mitochondrial damage. This study demonstrated that administration of crocetin to experimental animals during resuscitation from shock significantly improved postshock survival and reduced apoptosis. Crocetin is a component of saffron and has long been used in traditional medicine in Asia. METHODS Male Sprague-Dawley rats (350 ± 30 g) were randomly assigned to 1 of 4 groups of 8 animals. Hemorrhagic shock was induced by withdrawing blood until the mean arterial pressure was 35-40 mm Hg, and blood pressure was maintained at that level for 60 minutes with further withdrawals as needed. Resuscitation was carried out by administration of 21 mL/kg lactated Ringers solution and return of shed blood, with or without concurrent administration of crocetin (2 mg/kg). Control animals were sham-treated with surgical preparation, without shock or resuscitation, and with and without crocetin. Rats were sacrificed 24 hours after completion of resuscitation. The extent of activation of hepatic apoptosis was established by measuring levels of hepatic cytosolic cytochrome c, caspase-3, and bcl-2. A separate group of 53 animals treated identically was used to assess survival. RESULTS Crocetin administration during resuscitation resulted in less extensive activation of hepatic apoptosis and significantly increased survival relative to controls. CONCLUSIONS Crocetin administration to experimental animals during resuscitation post hemorrhage increased survival, at least in part by protecting the liver from activation of apoptotic cell death. This agent continues to show promise as a potential treatment strategy for hemorrhagic shock.