Structural Adaptation of the Excitation-Contraction Coupling Apparatus in Calsequestrin1-Null Mice during Postnatal Development.

The precise arrangement and peculiar interaction of transverse tubule (T-tubule) and sarcoplasmic reticulum (SR) membranes efficiently guarantee adequate contractile properties of skeletal muscle fibers. Fast muscle fibers from mice lacking calsequestrin 1 (CASQ1) are characterized by the profound ultrastructural remodeling of T-tubule/SR junctions. This study investigates the role of CASQ1, an essential component of calcium release units (CRUs), in the postnatal development of muscle fibers. By using CASQ1-knockout mice, we examined the maturation of CRUs and the involvement of different junctional proteins in the juxtaposition of the membrane system. Our morphological investigation of both wild-type (WT) and CASQ1-null extensor digitorum longus (EDL) fibers, from 1 week to 4 months of age, yielded noteworthy findings. Firstly, we observed that the absence of CASQ1 hindered the full maturation of CRUs, despite the correct localization of key junctional components (ryanodine receptor, dihydropyridine receptor, and triadin) to the junctional SR in adult animals. Furthermore, analysis of protein expression profiles related to T-tubule biogenesis and organization (junctophilin 1, amphiphysin 2, caveolin 3, and mitsugumin 29) demonstrated delayed progression in their expression during postnatal development in the absence of CASQ1, suggesting the impaired maturation of CRUs. The absence of CASQ1 directly impacts the proper assembly of CRUs during development and influences the expression and coordination of other proteins involved in T-tubule biogenesis and organization.

Evaluation of the Immunomodulatory Effects of a Probiotics and Natural Extract-Based Formulation in Bacterial-Induced Prostatitis.

Among the many factors inducing prostate inflammation, bacterial contribution is potentially underrated according to the scientific community. Bacterial prostatitis is characterized by modifications of the prostatic microenvironment, mainly driven by the immune system. Macrophages play a major role in bacterial prostatitis, secreting a plethora of proinflammatory and chemoattractive cytokines and proteolytic enzymes able to degrade the ECM, so facilitating the invasion of other immune cells. Consequently, macrophages represent a link between bacterial infection and prostate inflammation, as well as being the main target of prostate anti-inflammatory drugs and dietary supplements. This study aims to investigate the effect of a formulation composed of active principles and a probiotic strain with a particular focus on the anti-inflammatory effect in an in vitro bacterial prostatitis model. The results obtained showed that the formulation reduces the inflammatory response of prostatic epithelium induced by bacterial infection. This effect is mediated by the modulation of activated macrophages. Analysis of the cytokines released highlights that the tested formulation is able to reduce the expression of key proinflammatory cytokines involved in the pathogenesis of prostate diseases, in particular prostate cancer, and represents a valuable tool to prevent bacterial prostatitis and ensure favorable prostate health.

Efficacy Evaluation of Plant Products in the Treatment of Erectile Dysfunction Related to Diabetes.

Erectile dysfunction affects more than 50% of diabetic male patients, with a higher prevalence compared with the general population. Age, clinical factors, and lifestyle habits have been suggested to contribute to the pathophysiology and worsening of erectile dysfunction in diabetic patients. First- and second-line standard treatments are represented by phosphodiesterase type 5 (PDE5) inhibitors and alprostadil, respectively. However, natural compounds have been suggested to ameliorate this clinical condition. This study aims to preclinically characterize the potential synergism among plant-derived products for the improvement of erectile dysfunction in the diabetic condition. The effects of a nutritional supplement composed of Panax ginseng, Moringa oleifera and rutin, as single agents or as a mixture, were evaluated in a streptozotocin (STZ)-induced diabetic rat model with erectile dysfunction. The treatment efficacy was evaluated by measuring sexual-related parameters (i.e., mount and intromission latencies, the mount and intromission frequencies and the ejaculation latency). Results showed that only the mixture was able to significantly reduce the diabetes-related delay in mount latency (p < 0.01). Substantial similar effects were observed by measuring the intromission latency and the mean number of mounts was very similar between rats treated with the mixture and controls. Single agent treatments showed very low effects in terms of intromission frequency, whereas the mixture was able to increase this parameter. Additionally, a statistically significant reduced ejaculation latency was observed in rats treated with the mixture compared with the STZ control. These results are in agreement with the available literature and suggest that the study mixture may ameliorate sexual behavior compared with the administration of the study natural compounds as single agents in diabetic rats. Further preclinical and clinical studies are needed to perform a more comprehensive evaluation of the efficacy and safety of the study mixture.

PGC-1ß promotes enterocyte lifespan and tumorigenesis in the intestine.

The mucosa of the small intestine is renewed completely every 3-5 d throughout the entire lifetime by small populations of adult stem cells that are believed to reside in the bottom of the crypts and to migrate and differentiate into all the different populations of intestinal cells. When the cells reach the apex of the villi and are fully differentiated, they undergo cell death and are shed into the lumen. Reactive oxygen species (ROS) production is proportional to the electron transfer activity of the mitochondrial respiration chain. ROS homeostasis is maintained to control cell death and is finely tuned by an inducible antioxidant program. Here we show that peroxisome proliferator-activated receptor-γ coactivator-1ß (PGC-1ß) is highly expressed in the intestinal epithelium and possesses dual activity, stimulating mitochondrial biogenesis and oxygen consumption while inducing antioxidant enzymes. To study the role of PGC-1ß gain and loss of function in the gut, we generated both intestinal-specific PGC-1ß transgenic and PGC-1ß knockout mice. Mice overexpressing PGC-1ß present a peculiar intestinal morphology with very long villi resulting from increased enterocyte lifespan and also demonstrate greater tumor susceptibility, with increased tumor number and size when exposed to carcinogens. PGC-1ß knockout mice are protected from carcinogenesis. We show that PGC-1ß triggers mitochondrial respiration while protecting enterocytes from ROS-driven macromolecule damage and consequent apoptosis in both normal and dysplastic mucosa. Therefore, PGC-1ß in the gut acts as an adaptive self-point regulator, capable of providing a balance between enhanced mitochondrial activity and protection from increased ROS production.

Transcriptional regulation of the intestinal nuclear bile acid farnesoid X receptor (FXR) by the caudal-related homeobox 2 (CDX2).

Farnesoid X receptor (FXR, NR1H4) is a bile acid-activated transcription factor that belongs to the nuclear receptor superfamily. It is highly expressed in the enterohepatic system, where it senses bile acid levels to consequently reduce their synthesis while inducing their detoxification. Bile acids are intestinal tumor promoters and their concentrations have to be tightly regulated. Indeed, reduced expression of FXR in the intestine increases colorectal cancer susceptibility in mice, whereas its activation can promote apoptosis in genetically modified cells. Notably, despite the broad knowledge of the FXR enterohepatic transcriptional activity, the molecular mechanisms regulating FXR expression in the intestine are still unknown. Herein, by combining both gain and loss of function approaches and FXR promoter activity studies, we identified caudal-related homeobox 2 (CDX2) transcription factor as a positive regulator of FXR expression in the enterocytes. Our results provide a putative novel tool for modulating FXR expression against bile acid-related colorectal cancer progression.

Clustering nuclear receptors in liver regeneration identifies candidate modulators of hepatocyte proliferation and hepatocarcinoma.

BACKGROUND & AIMS: Liver regeneration (LR) is a valuable model for studying mechanisms modulating hepatocyte proliferation. Nuclear receptors (NRs) are key players in the control of cellular functions, being ideal modulators of hepatic proliferation and carcinogenesis. METHODS & RESULTS: We used a previously validated RT-qPCR platform to profile modifications in the expression of all 49 members of the NR superfamily in mouse liver during LR. Twenty-nine NR transcripts were significantly modified in their expression during LR, including fatty acid (peroxisome proliferator-activated receptors, PPARs) and oxysterol (liver X receptors, Lxrs) sensors, circadian masters RevErbα and RevErbß, glucocorticoid receptor (Gr) and constitutive androxane receptor (Car). In order to detect the NRs that better characterize proliferative status vs. proliferating liver, we used the novel Random Forest (RF) analysis to selected a trio of down-regulated NRs (thyroid receptor alpha, Trα; farsenoid X receptor beta, Fxrß; Pparδ) as best discriminators of the proliferating status. To validate our approach, we further studied PPARδ role in modulating hepatic proliferation. We first confirmed the suppression of PPARδ both in LR and human hepatocellular carcinoma at protein level, and then demonstrated that PPARδ agonist GW501516 reduces the proliferative potential of hepatoma cells. CONCLUSIONS: Our data suggest that NR transcriptome is modulated in proliferating liver and is a source of biomarkers and bona fide pharmacological targets for the management of liver disease affecting hepatocyte proliferation.

Microbiota modification with probiotics induces hepatic bile acid synthesis via downregulation of the Fxr-Fgf15 axis in mice.

Gut microbiota influences host health status by providing trophic, protective, and metabolic functions, including bile acid (BA) biotransformation. Microbial imprinting on BA signature modifies pool size and hydrophobicity, thus contributing to BA enterohepatic circulation. Microbiota-targeted therapies are now emerging as effective strategies for preventing and/or treating gut-related diseases. Here, we show that gut microbiota modulation induced by VSL#3 probiotics enhances BA deconjugation and fecal excretion in mice. These events are associated with changes in ileal BA absorption, repression of the enterohepatic farnesoid X receptor-fibroblast growth factor 15 (FXR-FGF15) axis, and increased hepatic BA neosynthesis. Treatment with a FXR agonist normalized fecal BA levels in probiotic-administered mice, whereas probiotic-induced alterations in BA metabolism are abolished upon FXR and FGF15 deficiency. Our data provide clear in vivo evidence that VSL#3 probiotics promote ileal BA deconjugation with subsequent fecal BA excretion and induce hepatic BA neosynthesis via downregulation of the gut-liver FXR-FGF15 axis.

Targeted therapy against chemoresistant colorectal cancers: Inhibition of p38α modulates the effect of cisplatin in vitro and in vivo through the tumor suppressor FoxO3A.

Chemoresistance is a major obstacle to effective therapy against colorectal cancer (CRC) and may lead to deadly consequences. The metabolism of CRC cells depends highly on the p38 MAPK pathway, whose involvement in maintaining a chemoresistant behavior is currently being investigated. Our previous studies revealed that p38α is the main p38 isoform in CRC cells. Here we show that p38α pharmacological inhibition combined with cisplatin administration decreases colony formation and viability of cancer cells and strongly increases Bax-dependent apoptotic cell death by activating the tumor suppressor protein FoxO3A. Our results indicate that FoxO3A activation up-regulates transcription of its target genes (p21, PTEN, Bim and GADD45), which forces both chemosensitive and chemoresistant CRC cells to undergo apoptosis. Additionally, we found that FoxO3A is required for apoptotic cell death induction, as confirmed by RNA interference experiments. In animal models xenografted with chemoresistant HT29 cells, we further confirmed that the p38-targeted dual therapy strategy produced an increase in apoptosis in cancer tissue leading to tumor regression. Our study uncovers a major role for the p38-FoxO3A axis in chemoresistance, thereby suggesting a new therapeutic approach for CRC treatment; moreover, our results indicate that Bax status may be used as a predictive biomarker.

Neuron-derived orphan receptor 1 promotes proliferation of quiescent hepatocytes.

BACKGROUND & AIMS: Studies of the transcriptional networks that regulate nuclear receptor-mediated proliferation of quiescent hepatocytes could lead to new information about liver growth and hepatoprotective strategies. METHODS: We used quantitative real-time PCR to analyze expression of neuron-derived orphan receptor 1 (Nor-1) and its target genes during liver regeneration after hepatectomy in mice, and in hepatocellular carcinoma (HCC) samples from patients. We used adenoviral vectors to express Nor-1 in normal liver (Ad/CMV/V5-Nor-1), or reduce its level with small hairpin RNAs (Ad/BLOCK-iT/Nor-1(small hairpin RNA)) after partial hepatectomy. RESULTS: Levels of Nor-1 messenger RNA and protein, and transcription of Nor-1 target genes (Ccnd1 and Vcam-1), increased during the late priming and proliferative phases of liver regeneration after partial hepatectomy. Levels of NOR-1 messenger RNA and transcription of its target gene CCND1 and of the NOR-1 subfamily member NUR-77 also increased in human HCC samples compared with paired HCC-free tissue. Ad-Nor-1(small hairpin RNA) reduced the hepatocyte proliferation after hepatectomy. Overexpression of Nor-1 in normal livers of mice induced proliferation of quiescent hepatocytes independently of interleukin-6 and tumor necrosis factor-α signaling. In gene expression profile analysis, Nor-1 altered expression of genes involved in the cell cycle, proliferation, and tumorigenesis. CONCLUSIONS: In mice, the orphan nuclear receptor Nor-1 activates proliferation of quiescent hepatocytes and is required for hepatocyte proliferation after partial hepatectomy. Nor-1 and its gene targets are also up-regulated in human HCC samples. Nor-1 activates a transcriptional program that induces hepatocyte proliferation independently of inflammatory signaling pathways.

Liver X receptors inhibit proliferation of human colorectal cancer cells and growth of intestinal tumors in mice.

BACKGROUND & AIMS: Liver X receptors (LXRs) are transcriptional regulators of cholesterol metabolism, controlling cholesterol flow into cells, catabolism, and efflux. Cholesterol controls cell proliferation; disruptions in cholesterol metabolism have been associated with the development of colon cancer. We investigated whether expression of activated LXR protects against intestinal tumorigenesis in mice. METHODS: We analyzed the development of colon cancer in mice that express a constitutive active form of LXRα only in the intestinal epithelium, under the control of villin promoter (iVP16LXRα). These mice were crossed with adenomatous polyposis coli (Apc)(min/+) mice, or given azoxymethane followed by dextran sodium sulfate, to assess intestinal tumor formation. We also assessed proliferation and apoptosis of a human colorectal cancer cell line (HT29) transfected with an adenoviral vector that expressed Ad VP16hLXRα, compared with cells expressing AdVP16 (control), and their ability to form xenograft tumors in mice. HT29 cells also were incubated with the LXR ligand GW3965. RESULTS: In human colorectal cancer cells, ligand-induced activation of LXR or transfection with Ad VP16hLXRα blocked the G1 phase, increased caspase-dependent apoptosis, and slowed growth of xenograft tumors in mice. iVP16LXRα mice formed fewer, smaller tumors than VP16 (control) mice after administration of azoxymethane and dextran sodium sulfate. APC(min/+)/iVP16LXRα mice also developed fewer, smaller intestinal tumors than APC(min/+)/iVP16 mice. Gene expression analysis indicated that activation of LXRα affected lipid metabolic networks and increased cholesterol efflux in the intestine. CONCLUSIONS: Expression of activated LXRα blocks proliferation of human colorectal cancer cells and slows the growth of xenograft tumors in mice. It also reduces intestinal tumor formation after administration of chemical carcinogens, and in Apc(min/+) mice. LXR agonists therefore might be developed as therapeutic treatments for colorectal cancer.

Hepatic-specific activation of peroxisome proliferator-activated receptor γ coactivator-1ß protects against steatohepatitis.

Development of hepatic steatosis and its progression to steatohepatitis may be the consequence of dysfunction of several metabolic pathways, such as triglyceride synthesis, very low-density lipoprotein (VLDL) secretion, and fatty acid ß-oxidation. Peroxisome proliferator-activated receptor γ coactivator-1ß (PGC-1ß) is a master regulator of mitochondrial biogenesis and oxidative metabolism, lipogenesis, and triglyceride (TG) secretion. Here we generated a novel mouse model with constitutive hepatic activation of PGC-1ß and studied the role of this transcriptional coactivator in dietary-induced steatosis and steatohepatitis. Selective activation of PGC-1ß within hepatocytes is able to protect the liver from lipid overload and from progression to fibrosis. The protective function exerted by PGC-1ß is due to its ability to induce mitochondrial oxidative phosphorylation, fatty acid ß-oxidation, and citrate cycle, as well as to decrease oxidative stress and promote TG secretion in the blood stream. These findings bolster the concept that a combined hepatic specific action of PGC-1ß on lipid synthesis and secretion, as well as on mitochondrial biogenesis and function, could protect against steatohepatitis.

A systems biology approach to the hepatic role of the oxysterol receptor LXR in the regulation of lipogenesis highlights a cross-talk with PPARα.

The Liver X Receptors (LXRs) α and ß and the Peroxisome Proliferator-Activated Receptor α (PPARα) are transcription factors that belong to class II nuclear receptors. They drive the expression of genes involved in hepatic lipid homeostasis and therefore are important targets for the prevention and treatment of nonalcoholic fatty liver disease (NAFLD). LXRs and PPARα are regulated by endogenous ligands, oxysterols and fatty acid derived molecules, respectively. In the liver, pharmacological activation of LXRs leads to the over-expression of genes involved in de novo lipogenesis, while PPARα is critical for fatty acid catabolism in nutrient deprivation. Even if these two nuclear receptors seemed to play opposite parts, recent studies have highlighted that PPARα also influence the expression of genes involved in fatty acids synthesis. In this study, we used pharmacological approaches and genetically engineered mice to investigate the cross-talk between LXRs and PPARα in the regulation of genes responsible for lipogenesis. We first investigated the effect of T0901317 and fenofibrate, two synthetic agonists of LXRs and PPARα, respectively. As expected, T0901317 and fenofibrate induce expression of genes involved LXR-dependent and PPARα-dependent lipogenic responses. Considering such overlapping effect, we then tested whether LXR agonist may influence PPARα driven response and vice versa. We show that the lack of PPARα does not influence the effects of T0901317 on lipogenic genes expression. However, PPARα deficiency prevents the up-regulation of genes involved in ω-hydroxylation that are induced by the LXR agonist. In addition, over-expression of lipogenic genes in response to fenofibrate is decreased in LXR knockout mice as well as the expression of PPARα target genes involved in fatty acid oxidation. Altogether, our work provides in vivo evidence for a central interconnection between nuclear receptors that drive hepatic lipid metabolism in response to oxysterol and fatty acids.

Sorafenib inhibits p38α activity in colorectal cancer cells and synergizes with the DFG-in inhibitor SB202190 to increase apoptotic response.

In the search for new strategies to efficiently fight colorectal cancer, efforts are being increasingly focused on targeting regulatory signaling pathways involved in cancer-specific features. As a result, several studies have recently addressed the therapeutic potential of molecularly-targeted drugs capable of inhibiting the activity of protein kinases involved in relevant signaling cascades. Here we show that simultaneous inhibition of the DFG-in and DFG-out conformations of p38α by means of type-I and type-II inhibitors is beneficial to impair more efficiently its kinase activity. Moreover, we found that SB202190 (type-I) and sorafenib (type-II) synergize at the molecular and biological level, as co-treatment with these compounds enhances tumor growth inhibition and induction of apoptosis both in colorectal cancer cell lines and animal models. These results support the need to reconsider sorafenib as a therapeutic agent against colorectal cancer and provide new insights that underline the importance to elucidate the activity of protein kinase inhibitors for the treatment of colorectal carcinoma.

Blocking p38/ERK crosstalk affects colorectal cancer growth by inducing apoptosis in vitro and in preclinical mouse models.

We recently demonstrated that p38α is required to maintain colorectal cancer (CRC) metabolism, as its inhibition leads to FoxO3A activation, autophagy, cell death, and tumor growth reduction both in vitro and in vivo. Here we show that inhibition of p38α is followed by TRAIL-mediated activation of caspase-8 and FoxO3A-dependent HER3 upregulation with consequent overactivation of the MEK-ERK1/2 survival pathway. p38α and MEK combined inhibition specifically induces apoptosis by enabling TRAIL signaling propagation through t-Bid and caspase-3, and fosters cell death in CRC cells and preclinical mouse models. Current MEK1-directed pharmacological strategies could thus be exploited, in combination with p38α inhibition, to develop new approaches for CRC treatment.

Selective activation of nuclear bile acid receptor FXR in the intestine protects mice against cholestasis.

BACKGROUND & AIMS: Cholestasis is a liver disorder characterized by impaired bile flow, reduction of bile acids (BAs) in the intestine, and retention of BAs in the liver. The farnesoid X receptor (FXR) is the transcriptional regulator of BA homeostasis. Activation of FXR by BAs reduces circulating BA levels in a feedback mechanism, repressing hepatic cholesterol 7α-hydroxylase (Cyp7a1), the rate-limiting enzyme for the conversion of cholesterol to BAs. This mechanism involves the hepatic nuclear receptor small heterodimer partner and the intestinal fibroblast growth factor (FGF) 19 and 15. We investigated the role of activation of intestine-specific FXR in reducing hepatic levels of BAs and protecting the liver from cholestasis in mice. METHODS: We generated transgenic mice that express a constitutively active FXR in the intestine. Using FXR gain- and loss-of-function models, we studied the roles of intestinal FXR in mice with intrahepatic and extrahepatic cholestasis. RESULTS: Selective activation of intestinal FXR induced FGF15 and repressed hepatic Cyp7a1, reducing the pool size of BAs and changing the BA pool composition. Activation of intestinal FXR protected mice from obstructive extrahepatic cholestasis after bile duct ligation or administration of α-naphthylisothiocyanate. In Mdr2(-/-) mice, transgenic expression of activated FXR in the intestine protected against liver damage, whereas absence of FXR promoted progression of liver disease. CONCLUSIONS: Activation of FXR transcription in the intestine protects the liver from cholestasis in mice by inducing FGF15 expression and reducing the hepatic pool of BA; this approach might be developed to reverse cholestasis in patients.

Bax is necessary for PGC1α pro-apoptotic effect in colorectal cancer cells.

We have recently shown that the transcriptional coactivator PGC1α, a master regulator of mitochondrial biogenesis and function, is involved in the control of the intestinal epithelium cell fate. Furthermore, PGC1α protects against colon cancer formation by promoting ROS accumulation and, consequently, mitochondria-mediated apoptosis. Here we provide an additional mechanistic insight into the tumor suppressor activity of PGC1α showing that its pro-apoptotic effect is mediated by Bax. In fact, PGC1α overexpression in HCT116 Bax (-/-) colorectal cancer cells stimulates mitochondrial production and activity, but it fails to induce cell death as well as to oppose tumor growth in the xenograft model. The lack of ROS accumulation in the Bax (-/-) cells strengthens our view that the PGC1α-induced oxidative burst represents one of the main apoptosis-driving factors in colorectal cancer cells.

Activation of bile salt nuclear receptor FXR is repressed by pro-inflammatory cytokines activating NF-κB signaling in the intestine.

UNLABELLED: Hyperactivation of NF-κB is a key factor in the pathophysiology of inflammatory bowel disease (IBD). We previously showed that the bile salt nuclear Farnesoid X Receptor (FXR) counter-regulates intestinal inflammation, possibly via repression of NF-κB. Here, we examine whether mutual antagonism between NF-κB and FXR exists. FXR and its target genes IBABP and FGF15/19 expression were determined in HT29 colon carcinoma cells and ex vivo in intestinal specimens of wild type (WT) and Fxr-ko mice, treated with/without FXR ligands (GW4064/INT-747) and inflammatory stimuli (TNFα/IL-1ß). In addition, FXR activation was studied in vivo in WT and Fxr-ko mice with DSS-colitis. The involvement of NF-κB in decreasing FXR activity was investigated by reporter assays and Glutathione S-transferase pulldown assays. FXR target gene expression was highly reduced by inflammatory stimuli in all model systems, while FXR mRNA expression was unaffected. In line with these results, reporter assays showed reduced FXR transcriptional activity upon TNFα/IL-1ß stimulation. We show that this reduction in FXR activity is probably mediated by NF-κB, since overexpression of NF-κB subunits p50 and/or p65 also lead to inhibition of FXR activity. Finally, we report that p65 and p50 physically interact with FXR in vitro. CONCLUSIONS: Together, these results indicate that intestinal inflammation strongly reduces FXR activation, probably via NF-κB-dependent tethering of FXR. Therefore, FXR not only inhibits inflammation, but also is targeted by the inflammatory response itself. This could result in a vicious cycle where reduced FXR activity results in less repression of inflammation, contributing to development of chronic intestinal inflammation. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.

Peroxisome proliferator-activated receptor-gamma coactivator 1-alpha (PGC1alpha) is a metabolic regulator of intestinal epithelial cell fate.

Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) is a transcriptional coactivator able to up-regulate mitochondrial biogenesis, respiratory capacity, oxidative phosphorylation, and fatty acid ß-oxidation with the final aim of providing a more efficient pathway for aerobic energy production. In the continuously renewed intestinal epithelium, proliferative cells in the crypts migrate along the villus axis and differentiate into mature enterocytes, increasing their respiratory capacity and finally undergoing apoptosis. Here we show that in the intestinal epithelial surface, PGC1α drives mitochondrial biogenesis and respiration in the presence of reduced antioxidant enzyme activities, thus determining the accumulation of reactive oxygen species and fostering the fate of enterocytes toward apoptosis. Combining gain- and loss-of-function genetic approaches in human cells and mouse models of intestinal cancer, we present an intriguing scenario whereby PGC1α regulates enterocyte cell fate and protects against tumorigenesis.

Farnesoid X receptor activation inhibits inflammation and preserves the intestinal barrier in inflammatory bowel disease.

BACKGROUND & AIMS: Inflammatory bowel disease (IBD) is characterised by chronic intestinal inflammation, resulting from dysregulation of the mucosal immune system and compromised intestinal epithelial barrier function. The bile salt, nuclear farnesoid X receptor (FXR), was recently implicated in intestinal antibacterial defence and barrier function. The aim of this study was to investigate the therapeutic potential of FXR agonists in the treatment of intestinal inflammation in complementary in vivo and in vitro models. METHODS: Colitis was induced in wild-type (WT) and Fxr-null mice using dextran sodium sulfate, and in WT mice using trinitrobenzenesulfonic acid. Mice were treated with vehicle or the FXR agonist INT-747, and colitis symptoms were assessed daily. Epithelial permeability assays and cytokine expression analysis were conducted in mouse colon and enterocyte-like cells (Caco-2/HT29) treated with medium or INT-747. Inflammatory cytokine secretion was determined by ELISA in various human immune cell types. RESULTS: INT-747-treated WT mice are protected from DSS- and TNBS-induced colitis, as shown by significant reduction of body weight loss, epithelial permeability, rectal bleeding, colonic shortening, ulceration, inflammatory cell infiltration and goblet cell loss. Furthermore, Fxr activation in intestines of WT mice and differentiated enterocyte-like cells downregulates expression of key proinflammatory cytokines and preserves epithelial barrier function. INT-747 significantly decreases tumour necrosis factor α secretion in activated human peripheral blood mononuclear cells, purified CD14 monocytes and dendritic cells, as well as in lamina propria mononuclear cells from patients with IBD. CONCLUSIONS: FXR activation prevents chemically induced intestinal inflammation, with improvement of colitis symptoms, inhibition of epithelial permeability, and reduced goblet cell loss. Furthermore, FXR activation inhibits proinflammatory cytokine production in vivo in the mouse colonic mucosa, and ex vivo in different immune cell populations. The findings provide a rationale to explore FXR agonists as a novel therapeutic strategy for IBD.

Characterizing Bile Acid and Lipid Metabolism in the Liver and Gastrointestinal Tract of Mice.

Mouse models that mimic human diseases are invaluable tools to study and discover genetic and pharmacological therapies for human diseases. The protocols described in this article are intended to assess general clinical parameters in the context of the enterohepatic system under both normal and pathological conditions. Methods are presented for characterizing liver and intestinal function with a focus on bile acid and lipid metabolism in the gut-liver axis. Curr. Protoc. Mouse Biol. 1:289-321 © 2011 by John Wiley & Sons, Inc.