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Teresa Esposito,1,2 Jean Marc Lobaccaro,3 Maria Grazia Esposito,4 Vincenzo Monda,1 Antonietta Messina,1 Giuseppe Paolisso,5 Bruno Varriale,2 Marcellino Monda,1 Giovanni Messina1,6 1Department of Experimental Medicine, Section of Human Physiology and Unit of Dietetics and Sports Medicine, 2Laboratory of Molecular Biology and Genetics, Department of Experimental Medicine, Second University of Naples, Naples, Italy; 3UMR, Clermont Université, Centre de Recherche en Nutrition Humaine d’Auvergne, Aubière Cedex, France; 4Complex Surgery Unit, Evangelic Hospital Villa Betania, 5Department of Scienze Mediche, Chirurgiche, Neurologiche, Metaboliche e dell’Invecchiamento, Second University of Naples, Naples, 6Department of Clinical and Experimental Medicine, University of Foggia, Foggia, Italy Abstract: The thyroid is one of the metabolism regulating glands. Its function is to determine the amount of calories that the body has to burn to maintain normal weight. Thyroiditides are inflammatory processes that mainly result in autoimmune diseases. We have conducted the present study in order to have a clear picture of both autoimmune status and the control of body weight. We have evaluated the amount of either thyroid hormones, or antithyroid, or anti-microsomal, or anti-peroxidase antibodies (Abs) in patients with high amounts of Abs. In a diet devoid of carbohydrates (bread, pasta, fruit, and rice), free from goitrogenic food, and based on body mass index, the distribution of body mass and intracellular and extracellular water conducted for 3 weeks gives the following results: patients treated as above showed a significant reduction of antithyroid (-40%, P0064). With regard to the body parameters measured in patients who followed this diet, reduction in body weight (-5%, P

Prostate cancer (PCa) is one of the most common cancers in men. Metastasis is the leading cause of death in prostate cancer patients. One of the crucial processes involved in metastatic spread is the "epithelial-mesenchymal transition" (EMT), which allows cells to acquire the ability to invade distant organs. Liver X Receptors (LXRs) are nuclear receptors that have been demonstrated to regulate EMT in various cancers, including hepatic cancer. Our study reveals that the LXR pathway can control pro-invasive cell capacities through EMT in prostate cancer, employing ex vivo and in vivo approaches. We characterized the EMT status of the commonly used LNCaP, DU145, and PC3 prostate cancer cell lines through molecular and immunohistochemistry experiments. The impact of LXR activation on EMT function was also assessed by analyzing the migration and invasion of these cell lines in the absence or presence of an LXR agonist. Using in vivo experiments involving NSG-immunodeficient mice xenografted with PC3-GFP cells, we were able to study metastatic spread and the effect of LXRs on this process. LXR activation led to an increase in the accumulation of Vimentin and Amphiregulin in PC3. Furthermore, the migration of PC3 cells significantly increased in the presence of the LXR agonist, correlating with an upregulation of EMT. Interestingly, LXR activation significantly increased metastatic spread in an NSG mouse model. Overall, this work identifies a promoting effect of LXRs on EMT in the PC3 model of advanced prostate cancer.

Nonalcoholic fatty liver disease (NAFLD) is defined by a set of hepatic conditions ranging from steatosis to steatohepatitis (NASH), characterized by inflammation and fibrosis, eventually predisposing to hepatocellular carcinoma (HCC). Together with fatty acids (FAs) originated from adipose lipolysis and hepatic lipogenesis, intestinal-derived FAs are major contributors of steatosis. However, the role of mono-unsaturated FAs (MUFAs) in NAFLD development is still debated. We previously established the intestinal capacity to produce MUFAs, but its consequences in hepatic functions are still unknown. Here, we aimed to determine the role of the intestinal MUFA-synthetizing enzyme stearoyl-CoA desaturase 1 (SCD1) in NAFLD. We used intestinal-specific Scd1-KO (iScd1 -/- ) mice and studied hepatic dysfunction in different models of steatosis, NASH, and HCC. Intestinal-specific Scd1 deletion decreased hepatic MUFA proportion. Compared with controls, iScd1 -/- mice displayed increased hepatic triglyceride accumulation and derangement in cholesterol homeostasis when fed a MUFA-deprived diet. Then, on Western diet feeding, iScd1 -/- mice triggered inflammation and fibrosis compared with their wild-type littermates. Finally, intestinal-Scd1 deletion predisposed mice to liver cancer. Conclusions: Collectively, these results highlight the major importance of intestinal MUFA metabolism in maintaining hepatic functions and show that gut-derived MUFAs are protective from NASH and HCC., (© 2022 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Prostate cancer is the most common cancer in aging men. Despite recent progress, there are still few effective treatments to cure its aggressive and metastatic stages. A better understanding of the molecular mechanisms driving disease initiation and progression appears essential to support the development of more efficient therapies and improve patient care. To do so, multiple research models, such as cell culture and mouse models, have been developed over the years and have improved our comprehension of the biology of the disease. Recently, a new model has been added with the use of the Drosophila accessory gland. With a high level of conservation of major signaling pathways implicated in human disease, this functional equivalent of the prostate represents a powerful, inexpensive, and rapid in vivo model to study epithelial carcinogenesis. The purpose of this review is to quickly overview the existing prostate cancer models, including their strengths and limitations. In particular, we discuss how the Drosophila accessory gland can be integrated as a convenient complementary model by bringing new understanding in the mechanisms driving prostate epithelial tumorigenesis, from initiation to metastatic formation.

Background & Aims: The enzyme stearoyl-coenzyme A desaturase 1 (SCD or SCD1) produces monounsaturated fatty acids by introducing double bonds into saturated bonds between carbons 9 and 10, with oleic acid as the main product. SCD1 is present in the intestinal epithelium, and fatty acids regulate cell proliferation, so we investigated the effects of SCD1-induced production of oleic acid in enterocytes in mice., Methods: We generated mice with disruption of Scd1 selectively in the intestinal epithelium (iScd1 -/- mice) on a C57BL/6 background; iScd1 +/+ mice were used as controls. We also generated iScd1 -/- Apc Min/+ mice and studied cancer susceptibility. Mice were fed a chow, oleic acid-deficient, or oleic acid-rich diet. Intestinal tissues were collected and analyzed by histology, reverse transcription quantitative polymerase chain reaction, immunohistochemistry, and mass spectrometry, and tumors were quantified and measured., Results: Compared with control mice, the ileal mucosa of iScd1 -/- mice had a lower proportion of palmitoleic (C16:1 n-7) and oleic acids (C18:1 n-9), with accumulation of stearic acid (C18:0); this resulted a reduction of the Δ9 desaturation ratio between monounsaturated (C16:1 n-7 and C18:1 n-9) and saturated (C16:0 and C18:0) fatty acids. Ileal tissues from iScd1 -/- mice had increased expression of markers of inflammation activation and crypt proliferative genes compared with control mice. The iScd1 -/- Apc Min/+ mice developed more and larger tumors than iScd1 +/+ Apc Min/+ mice. iScd1 -/- Apc Min/+ mice fed the oleic acid-rich diet had reduced intestinal inflammation and significantly lower tumor burden compared with mice fed a chow diet., Conclusions: In studies of mice, we found intestinal SCD1 to be required for synthesis of oleate in the enterocytes and maintenance of fatty acid homeostasis. Dietary supplementation with oleic acid reduces intestinal inflammation and tumor development in mice., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The extraction of RNA and lipids from a large number of biological samples is time-consuming and costly with steps required for both transcriptomic and lipidomic approaches. Most protocols rely on independent extraction of nucleic acids and lipids from a single sample, thereby increasing the need for biological material and inducing variability in data analysis. We investigated whether it is possible to use a standard RNA extraction procedure to analyze not only RNA levels, but also lipids in a single liver sample. We show that the organic phase obtained when using standard reagents for RNA extraction can be used to analyze lipids, including neutral lipids and fatty acids, by gas chromatography. We applied this technique to an analysis of lipids and the associated gene expression pattern in mice with hepatic steatosis induced by pharmacological activation of nuclear receptor LXR.

Dendrogenin A (DDA) is a newly discovered cholesterol metabolite with tumor suppressor properties. Here, we explored its efficacy and mechanism of cell death in melanoma and acute myeloid leukemia (AML). We found that DDA induced lethal autophagy in vitro and in vivo, including primary AML patient samples, independently of melanoma Braf status or AML molecular and cytogenetic classifications. DDA is a partial agonist on liver-X-receptor (LXR) increasing Nur77, Nor1, and LC3 expression leading to autolysosome formation. Moreover, DDA inhibited the cholesterol biosynthesizing enzyme 3β-hydroxysterol-Δ 8,7 -isomerase (D8D7I) leading to sterol accumulation and cooperating in autophagy induction. This mechanism of death was not observed with other LXR ligands or D8D7I inhibitors establishing DDA selectivity. The potent anti-tumor activity of DDA, its original mechanism of action and its low toxicity support its clinical evaluation. More generally, this study reveals that DDA can direct control a nuclear receptor to trigger lethal autophagy in cancers.

Olive oil consumption is beneficial for health as it is associated with a decreased prevalence of cancer and cardiovascular diseases. Oleic acid is, by far, the most abundant component of olive oil. Since it can be made through de novo synthesis in animals, it is not an essential fatty acid. While it has become clear that dietary oleic acid regulates many biological processes, the signaling pathway involved in these regulations remains poorly defined. In this work we tested the impact of an oleic acid-rich diet on hepatic gene expression. We were particularly interested in addressing the contribution of Liver X Receptors (LXR) in the control of genes involved in hepatic lipogenesis, an essential process in whole body energy homeostasis. We used wild-type mice and transgenic mice deficient for both α and β Liver X Receptor isoforms (LXR-/-) fed a control or an oleate enriched diet. We observed that hepatic-lipid accumulation was enhanced as well as the expression of lipogenic genes in the liver of wild-type mice fed the oleate enriched diet. In contrast, none of these changes occurred in the liver of LXR-/- mice. Strikingly, oleate-rich diet reduced cholesterolemia in wild-type mice and induced signs of liver inflammation and damage in LXR-/- mice but not in wild-type mice. This work suggests that dietary oleic acid reduces cholesterolemia while promoting LXR-dependent hepatic lipogenesis without detrimental effects to the liver.

Liver X receptors (LXRs) are key regulators of lipid homeostasis and are involved in multiple testicular functions. The Lxrα(-/-);Lxrβ(-/-) mice have illuminated the roles of both isoforms in maintenance of the epithelium in the seminiferous tubules, spermatogenesis, and T production. The requirement for LXRβ in Sertoli cells have been emphasized by early abnormal cholesteryl ester accumulation in the Lxrβ(-/-) and Lxrα(-/-);Lxrβ(-/-) mice. Other phenotypes, such as germ cell loss and hypogonadism, occur later in life in the Lxrα(-/-);Lxrβ(-/-) mice. Thus, LXRβ expression in Sertoli cells seems to be essential for normal testicular physiology. To decipher the roles of LXRβ within the Sertoli cells, we generated Lxrα(-/-);Lxrβ(-/-):AMH-Lxrβ transgenic mice, which reexpress Lxrβ in Sertoli cells in the context of Lxrα(-/-);Lxrβ(-/-) mice. In addition to lipid homeostasis, LXRβ is necessary for maintaining the blood-testis barrier and the integrity of the germ cell epithelium. LXRβ is also implicated in the paracrine action of Sertoli cells on Leydig cells to modulate T synthesis. The Lxrα(-/-);Lxrβ(-/-) and Lxrα(-/-);Lxrβ(-/-):AMH-Lxrβ mice exhibit lipid accumulation in germ cells after the Abcg8 down-regulation, suggesting an intricate LXRβ-dependent cooperation between the Sertoli cells and germ cells to ensure spermiogenesis. Further analysis revealed also peritubular smooth muscle defects (abnormal lipid accumulation and disorganized smooth muscle actin) and spermatozoa stagnation in the seminiferous tubules. Together the present work elucidates specific roles of LXRβ in Sertoli cell physiology in vivo beyond lipid homeostasis.

Objective: Liver X receptors (LXRs) modulate cholesterol and fatty acid homeostasis as well as inflammation. This study aims to decipher the role of LXRs in the regulation of polyunsaturated fatty acid (PUFA) synthesis in macrophages in the context of atherosclerosis., Approach and Results: Transcriptomic analysis in human monocytes and macrophages was used to identify putative LXR target genes among enzymes involved in PUFA biosynthesis. In parallel, the consequences of LXR activation or LXR invalidation on PUFA synthesis and distribution were determined. Finally, we investigated the impact of LXR activation on PUFA metabolism in vivo in apolipoprotein E-deficient mice. mRNA levels of acyl-CoA synthase long-chain family member 3, fatty acid desaturases 1 and 2, and fatty acid elongase 5 were significantly increased in human macrophages after LXR agonist treatment, involving both direct and sterol responsive element binding protein-1-dependent mechanisms. Subsequently, pharmacological LXR agonist increased long chain PUFA synthesis and enhanced arachidonic acid content in the phospholipids of human macrophages. Increased fatty acid desaturases 1 and 2 and acyl-CoA synthase long-chain family member 3 mRNA levels as well as increased arachidonic acid to linoleic acid and docosahexaenoic acid to eicosapentaenoic acid ratios were also found in atheroma plaque and peritoneal foam cells from LXR agonist-treated mice. By contrast, murine LXR-deficient macrophages displayed reduced expression of fatty acid elongase 5, acyl-CoA synthase long-chain family member 3 and fatty acid desaturases 1, as well as decreased cellular levels of docosahexaenoic acid and arachidonic acid., Conclusions: Our results indicate that LXR activation triggers PUFA synthesis in macrophages, which results in significant alterations in the macrophage lipid composition. Moreover, we demonstrate here that LXR agonist treatment modulates PUFA metabolism in atherosclerotic arteries., (© 2015 American Heart Association, Inc.)

The small heterodimer partner (SHP, nuclear receptor subfamily 0, group B, member 2; NR0B2) is an atypical nuclear receptor known mainly for its role in bile acid homeostasis in the enterohepatic tract. We previously showed that NR0B2 controls testicular functions such as testosterone synthesis. Moreover, NR0B2 mediates the deleterious testicular effects of estrogenic endocrine disruptors leading to infertility. The endocrine homeostasis is essential for health, because it controls many physiological functions. This is supported by a large number of studies demonstrating that alterations of steroid activity lead to several kinds of diseases such as obesity and infertility. Within the testis, the functions of the Leydig cells are mainly controlled by the hypothalamo-pituitary axis via LH/chorionic gonadotropin (CG). Here, we show that LH/CG represses Nr0b2 expression through the protein kinase A-AMP protein kinase pathway. Moreover, using a transgenic mouse model invalidated for Nr0b2, we point out that NR0B2 mediates the repression of testosterone synthesis and subsequent germ cell apoptosis induced by exposure to anti-GnRH compound. Together, our data demonstrate a new link between hypothalamo-pituitary axis and NR0B2 in testicular androgen metabolism, making NR0B2 a major actor of testicular physiology in case of alteration of LH/CG levels.

Essential oils are widely used in pharmaceutical, sanitary, cosmetic, agriculture and food industries for their bactericidal, virucidal, fungicidal, antiparasitical and insecticidal properties. Their anticancer activity is well documented. Over a hundred essential oils from more than twenty plant families have been tested on more than twenty types of cancers in last past ten years. This review is focused on the activity of essential oils and their components on various types of cancers. For some of them the mechanisms involved in their anticancer activities have been carried out.

Objective: To determine whether the transcription factors liver X receptors (LXRs) and their downstream genes, which are involved in the regulation of several testicular functions in mouse models, are differentially expressed in testes of men with nonobstructive azoospermia (NOA) or obstructive azoospermia (OA)., Design: Prospective study., Setting: University hospital., Patient(s): Patients with various types of NOA (n=22) and with OA (n=5)., Intervention(s): Human testicular biopsies., Main Outcome Measure(s): Transcript levels were measured in testicular biopsies with the use of quantitative polymerase chain reaction. Correlations of LXR mRNA levels with the number of germ cells, the expression of proliferation and apoptosis markers, and the amount of intratesticular lipids and testosterone were evaluated. The localization of LXRα was analyzed by immunofluorescence., Result(s): LXR mRNA levels were decreased by 49%-98% in NOA specimens and positively correlated with germ cell number. Accumulations of IDOL and SREBP1c (LXR targets involved in lipid homeostasis) were 1.8-2.1 times lower in NOA samples and mRNA levels of the SREBP1c target gene ELOVL6 were increased 1.9-2.4-fold. Interestingly, the amount of triglycerides and free fatty acids were higher in NOA testes (3.4-12.2-fold). LXRα was present in Leydig cells. Accumulations of LXR downstream genes encoding the steroidogenic proteins StAR and 3βHSD2 were higher in NOA testes (5.9-12.8-fold)., Conclusion(s): Knowledge of changes in the transcript levels of LXRs and some of their downstream genes during altered spermatogenesis may help us to better understand the physiopathology of testicular failure in azoospermic patients., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

This research highlights the chemical composition, antioxidant, anti-inflammatory and anti-proliferative activities of essential oils from leaves of Ocimum basilicum, Ocimum americanum, Hyptis spicigera, Lippia multiflora, Ageratum conyzoides, Eucalyptus camaldulensis and Zingiber officinale. Essential oils were analyzed by gas chromatography-mass spectrometry and gas chromatography-flame ionization detector. Major constituents were α-terpineol (59.78%) and β-caryophyllene (10.54%) for Ocimum basilicum; 1, 8-cineol (31.22%), camphor (12.730%), α-pinene (6.87%) and trans α-bergamotene (5.32%) for Ocimum americanum; β-caryophyllene (21%), α-pinene (20.11%), sabinene (10.26%), β-pinene (9.22%) and α-phellandrene (7.03%) for Hyptis spicigera; p-cymene (25.27%), β-caryophyllene (12.70%), thymol (11.88), γ-terpinene (9.17%) and thymyle acetate (7.64%) for Lippia multiflora; precocene (82.10%)for Ageratum conyzoides; eucalyptol (59.55%), α-pinene (9.17%) and limonene (8.76%) for Eucalyptus camaldulensis; arcurcumene (16.67%), camphene (12.70%), zingiberene (8.40%), β-bisabolene (7.83%) and β-sesquiphellandrène (5.34%) for Zingiber officinale. Antioxidant activities were examined using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) methods. O. basilicum and L. multiflora exhibited the highest antioxidant activity in DPPH and ABTS tests, respectively. Anti-inflammatory properties were evaluated by measuring the inhibition of lipoxygenase activity and essential oil of Z. officinale was the most active. Anti-proliferative effect was assayed by the measurement of MTT on LNCaP and PC-3 prostate cancer cell lines, and SF-763 and SF-767 glioblastoma cell lines. Essential oils from A. conyzoides and L. multiflora were the most active on LNCaP and PC-3 cell lines, respectively. The SF-767 glioblastoma cell line was the most sensitive to O. basilicum and L. multiflora EOs while essential oil of A. conyzoides showed the highest activity on SF-763 cells. Altogether these results justify the use of these plants in traditional medicine in Burkina Faso and open a new field of investigation in the characterization of the molecules involved in anti-proliferative processes.

Bile acids are cholesterol metabolites that have been extensively studied in recent decades. In addition to having ancestral roles in digestion and fat solubilization, bile acids have recently been described as signaling molecules involved in many physiological functions, such as glucose and energy metabolisms. These signaling pathways involve the activation of the nuclear receptor farnesoid X receptor (FXRα) or of the G protein-coupled receptor TGR5. In this review, we will focus on the emerging role of FXRα, suggesting important functions for the receptor in steroid metabolism. It has been described that FXRα is expressed in the adrenal glands and testes, where it seems to control steroid production. FXRα also participates in steroid catabolism in the liver and interferes with the steroid signaling pathways in target tissues via crosstalk with steroid receptors. In this review, we discuss the potential impacts of bile acid (BA), through its interactions with steroid metabolism, on glucose metabolism, sexual function, and prostate and breast cancers. Although several of the published reports rely on in vitro studies, they highlight the need to understand the interactions that may affect health. This effect is important because BA levels are increased in several pathophysiological conditions related to liver injuries. Additionally, BA receptors are targeted clinically using therapeutics to treat liver diseases, diabetes, and cancers.

LXR (Liver X Receptors) act as "sensor" proteins that regulate cholesterol uptake, storage, and efflux. LXR signaling is known to influence proliferation of different cell types including human prostatic carcinoma (PCa) cell lines. This study shows that deletion of LXR in mouse fed a high-cholesterol diet recapitulates initial steps of PCa development. Elevation of circulating cholesterol in Lxrαβ-/- double knockout mice results in aberrant cholesterol ester accumulation and prostatic intra-epithelial neoplasia. This phenotype is linked to increased expression of the histone methyl transferase EZH2 (Enhancer of Zeste Homolog 2), which results in the down-regulation of the tumor suppressors Msmb and Nkx3.1 through increased methylation of lysine 27 of histone H3 (H3K27) on their promoter regions. Altogether, our data provide a novel link between LXR, cholesterol homeostasis, and epigenetic control of tumor suppressor gene expression., Competing Interests: The authors have declared that no competing interests exist.

Recent studies underline the implication of Liver X Receptors (LXRs) in several prostate diseases such as benign prostatic hyperplasia (BPH) and prostate cancer. In order to understand the molecular mechanisms involved, we derived epithelial cells from dorsal prostate (MPECs) of wild type (WT) or Lxrαβ-/- mice. In the WT MPECs, our results show that LXR activation reduces proliferation and correlates with the modification of the AKT-survival pathway. Moreover, LXRs regulate lipid homeostasis with the regulation of Abca1, Abcg1 and Idol, and, in a lesser extent, Srebp1, Fas and Acc. Conversely cells derived from Lxrαβ-/- mice show a higher basal phosphorylation and consequently activation of the survival/proliferation transduction pathways AKT and MAPK. Altogether, our data point out that the cell model we developed allows deciphering the molecular mechanisms inducing the cell cycle arrest. Besides, we show that activated LXRs regulate AKT and MAPK transduction pathways and demonstrate that LXRs could be good pharmacological targets in prostate disease such as cancer.

Cholesterol is a fundamental molecule for life. Located in the cell membrane, this sterol participates to the cell signaling of growth factors. Inside the cell it can be converted in hormones such as androgens or modulate the immune response. Such important functions could not be solely dependent of external supply by diet hence de novo synthesis could occur from acetate in almost all mammalian cells. If a deficiency in cholesterol sourcing leads to development troubles, overstocking has been associated to various diseases such as atherosclerosis and cancers. Cholesterol homeostasis should thus be tightly regulated at the uptake, de novo synthesis, storage and export processes. Various transcription factors have been described these last years as important to regulate cholesterol levels. Besides, synthetic molecules have been developed for many years to modulate cholesterol synthesis, such as statins. Many articles have associated prostate cancer, whose incidence is constantly increasing, to cholesterol disequilibrium. Targeting cholesterol could thus be a new pharmacological hit to counteract the initiation, development and/or progression of prostate cancer. Among the transcription factors regulating cholesterol homeostasis, the nuclear receptors Liver X Receptors (LXRs) control cholesterol uptake and export. Targeting the LXRs offers a new field of investigation to treat cancer. This review highlights the molecular relationships among LXRs, prostate cancer and cholesterol and why LXRs have good chance to be targeted one day in this tumor. LXRs, prostate cancer and cholesterol, more than a "Ménage à trois", The Good, the Bad and the Ugly.

In the last decades, studies in rodents have highlighted links between in utero and/or neonatal exposures to molecules that alter endocrine functions and the development of genital tract abnormalities, such as cryptorchidism, hypospadias, and impaired spermatogenesis. Most of these molecules, called endocrine disrupters exert estrogenic and/or antiandrogenic activities. These data led to the hypothesis of the testicular dysgenesis syndrome which postulates that these disorders are one clinical entity and are linked by epidemiological and pathophysiological relations. Furthermore, infertility has been stated as a risk factor for testicular cancer (TC). The incidence of TC has been increasing over the past decade. Most of testicular germ cell cancers develop through a pre-invasive carcinoma in situ from fetal germ cells (primordial germ cell or gonocyte). During their development, fetal germ cells undergo epigenetic modifications. Interestingly, several lines of evidence have shown that gene regulation through epigenetic mechanisms (DNA and histone modifications) plays an important role in normal development as well as in various diseases, including TC. Here we will review chromatin modifications which can affect testicular physiology leading to the development of TC; and highlight potential molecular pathways involved in these alterations in the context of environmental exposures.

Benign prostatic hyperplasia is a nonmalignant enlargement of the prostate that commonly occurs in older men. We show that liver X receptor (Lxr)-α knockout mice (lxrα(-/-)) develop ventral prostate hypertrophy, correlating with an overaccumulation of secreted proteins in prostatic ducts and an alteration of vesicular trafficking in epithelial cells. In the fluid of the lxrα(-/-) prostates, spermine binding protein is highly accumulated and shows a 3000-fold increase of its mRNA. This overexpression is mediated by androgen hypersensitivity in lxrα(-/-) mice, restricted to the ventral prostate. Generation of chimeric recombinant prostates demonstrates that Lxrα is involved in the establishment of the epithelial-mesenchymal interactions in the mouse prostate. Altogether these results point out the crucial role of Lxrα in the homeostasis of the ventral prostate and suggest lxrα(-/-) mice may be a good model to investigate the molecular mechanisms of benign prostatic hyperplasia.

Oxysterols are reactive molecules generated from the oxidation of cholesterol. Their implication in cholesterol homeostasis and in the progression of neurodegenerative disorders is well known, but few data are available for their functions in the peripheral nervous system. Our aim was to study the influence of oxysterols on myelin gene expression and myelin sheath formation in peripheral nerves. We show by gas chromatography/mass spectrometry that Schwann cells and sciatic nerves contain 24(S)-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol and that they express their biosynthetic enzymes and receptors (liver X receptors LXRα and LXRβ). We demonstrate that oxysterols inhibit peripheral myelin gene expression [myelin protein zero (MPZ) and peripheral myelin protein-22 (PMP22)] in a Schwann cell line. This downregulation is mediated by either LXRα or LXRβ, depending on the promoter context, as suggested by siRNA strategy and chromatin immunoprecipitation assays in Schwann cells and in the sciatic nerve of LXR knock-out mice. Importantly, the knock-out of LXR in mice results in thinner myelin sheaths surrounding the axons. Oxysterols repress myelin genes via two mechanisms: by binding of LXRs to myelin gene promoters and by inhibiting the Wnt/β-catenin pathway that is crucial for the expression of myelin genes. The Wnt signaling components (Disheveled, TCF/LEF, β-catenin) are strongly repressed by oxysterols. Furthermore, the recruitment of β-catenin at the levels of the MPZ and PMP22 promoters is decreased. Our data reveal new endogenous mechanisms for the negative regulation of myelin gene expression, highlight the importance of oxysterols and LXR in peripheral nerve myelination, and open new perspectives of treating demyelinating diseases with LXR agonists.

This work shows that an overload of dietary cholesterol causes complete infertility in dyslipidemic male mice (the Liver X Receptor-deficient mouse model). Infertility resulted from post-testicular defects affecting the fertilizing potential of spermatozoa. Spermatozoa of cholesterol-fed lxr-/- animals were found to be dramatically less viable and motile, and highly susceptible to undergo a premature acrosome reaction. We also provide evidence, that this lipid-induced infertility is associated with the accelerated appearance of a highly regionalized epididymal phenotype in segments 1 and 2 of the caput epididymidis that was otherwise only observed in aged LXR-deficient males. The epididymal epithelial phenotype is characterized by peritubular accumulation of cholesteryl ester lipid droplets in smooth muscle cells lining the epididymal duct, leading to their transdifferentiation into foam cells that eventually migrate through the duct wall, a situation that resembles the inflammatory atherosclerotic process. These findings establish the high level of susceptibility of epididymal sperm maturation to dietary cholesterol overload and could partly explain reproductive failures encountered by young dyslipidemic men as well as ageing males wishing to reproduce.

Thyroid hormone (TR) and liver X (LXR) receptors are transcription factors involved in lipogenesis. Both receptors recognize the same consensus DNA-response element in vitro. It was previously shown that their signaling pathways interact in the control of cholesterol elimination in the liver. In the present study, carbohydrate-response element-binding protein (ChREBP), a major transcription factor controlling the activation of glucose-induced lipogenesis in liver, is characterized as a direct target of thyroid hormones (TH) in liver and white adipose tissue (WAT), the two main lipogenic tissues in mice. Using genetic and molecular approaches, ChREBP is shown to be specifically regulated by TRbeta but not by TRalpha in vivo, even in WAT where both TR isoforms are expressed. However, this isotype specificity is not found in vitro. This TRbeta specific regulation correlates with the loss of TH-induced lipogenesis in TRbeta(-/-) mice. Fasting/refeeding experiments show that TRbeta is not required for the activation of ChREBP expression particularly marked in WAT following refeeding. However, TH can stimulate ChREBP expression in WAT even under fasting conditions, suggesting completely independent pathways. Because ChREBP has been described as an LXR target, the interaction of LXR and TRbeta in ChREBP regulation was assayed both in vitro and in vivo. Each receptor recognizes a different response element on the ChREBP promoter, located only 8 bp apart. There is a cross-talk between LXR and TRbeta signaling on the ChREBP promoter in liver but not in WAT where LXR does not regulate ChREBP expression. The molecular basis for this cross-talk has been determined in in vitro systems.

Background: Prospective studies indicate that tomato consumers are protected against prostate cancer. Lycopene has been hypothesized to be responsible for tomato health benefits., Objective: Our aim was to differentiate the effects of tomato matrix from those of lycopene by using lycopene-rich red tomatoes, lycopene-free yellow tomatoes, and purified lycopene., Design: Thirty healthy men (aged 50-70 y old) were randomly assigned to 2 groups after a 2-wk washout period. In a crossover design, each group consumed yellow and red tomato paste (200 g/d, which provided 0 and 16 mg lycopene, respectively) as part of their regular diet for 1 wk separated by 2 wk of washout. Then, in a parallel design, the first group underwent supplementation with purified lycopene (16 mg/d) for 1 wk, whereas the second group received a placebo. Sera collected before and after the interventions were incubated with lymph node cancer prostate cells to measure the expression of 45 target genes., Results: Circulating lycopene concentration increased only after consumption of red tomato paste and purified lycopene. Lipid profile, antioxidant status, prostate-specific antigen, and insulin-like growth factor I were not modified by consumption of tomato pastes and lycopene. We observed significant up-regulation of IGFBP-3 and Bax:Bcl-2 ratio and down-regulation of cyclin-D1, p53, and Nrf-2 after cell incubation with sera from men who consumed red tomato paste when compared with sera collected after the first washout period, with intermediate values for yellow tomato paste consumption. Cell incubation with sera from men who consumed purified lycopene led to significant up-regulation of IGFBP-3, c-fos, and uPAR compared with sera collected after placebo consumption., Conclusion: Dietary lycopene can affect gene expression whether or not it is included in its food matrix. This trial was registered by the French Health Ministry at http://www.sante-sports.gouv.fr as 2006-A00396-45.

Cholesterol is a structural component of lipid rafts within the plasma membrane. These domains, used as platforms for various signaling molecules, regulate cellular processes including cell survival. Cholesterol contents are tightly correlated with the structure and function of lipid rafts. Liver X receptors (LXRs) have a central role in the regulation of cholesterol homeostasis within the cell. Therefore, we investigated whether these nuclear receptors could modulate lipid raft signaling and consequently alter prostate cancer (PCa) cell survival. Treatment with the synthetic LXR agonist T0901317 downregulated the AKT survival pathway and thus induced apoptosis of LNCaP PCa cells in both xenografted nude mice and cell culture. The decrease in tumor cholesterol content resulted from the upregulation of ABCG1 and the subsequent increase in reverse cholesterol transport. RNA interference experiments showed that these effects were mediated by LXRs. Atomic force microscopy scanning of the inner plasma membrane sheet showed smaller and thinner lipid rafts after LXR stimulation, associated with the downregulation of AKT phosphorylation in these lipid rafts. Replenishment of cell membranes with exogenous cholesterol antagonized these effects, showing that cholesterol is a key modulator in this process. Altogether, pharmacological modulation of LXR activity could thus reduce prostate tumor growth by enhancing apoptosis in a lipid raft-dependent manner.

Mammalian spermatozoa undergo important plasma membrane maturation steps during epididymal transit. Among these, changes in lipids and cholesterol are of particular interest as they are necessary for fertilization. However, molecular mechanisms regulating these transformations inside the epididymis are still poorly understood. Liver X receptors (LXRs), the nuclear receptors for oxysterols, are of major importance in intracellular cholesterol homeostasis, and LXR(-/-)-deficient male mice have already been shown to have reduced fertility at an age of 5 months and complete sterility for 9-month-old animals. This sterility phenotype is associated with testes and caput epididymides epithelial defects. The research presented here was aimed at investigating how LXRs act in the male caput epididymidis by analyzing key actors in cholesterol homeostasis. We show that accumulation of cholesteryl esters in LXR(-/-) male mice is associated with a specific loss of ABCA1 and an increase in apoptosis of apical cells of the proximal caput epididymidis. ATP-binding cassette G1 (ABCG1) and scavenger receptor B1 (SR-B1), two other cholesterol transporters, show little if any modifications. Our study also revealed that SR-B1 appears to have a peculiar expression pattern along the epididymal duct. These results should help in understanding the functional roles of LXR in cholesterol trafficking processes in caput epididymidis.

Rationale: Liver X receptors (LXRs) are oxysterol-activated nuclear receptors that are involved in the control of cholesterol homeostasis and inflammatory response. Human monocytes and macrophages express high levels of these receptors and are appropriate cells to study the response to LXR agonists., Objective: The purpose of this study was to identify new LXR targets in human primary monocytes and macrophages and the consequences of their activation., Methods and Results: We show that LXR agonists significantly increase the mRNA and protein levels of the retinoic acid receptor (RAR)alpha in primary monocytes and macrophages. LXR agonists promote RARalpha gene transcription through binding to a specific LXR response element on RARalpha gene promoter. Preincubation of monocytes or macrophages with LXR agonists before RARalpha agonist treatment enhances synergistically the expression of several RARalpha target genes. One of these genes encodes transglutaminase (TGM)2, a key factor required for macrophage phagocytosis. Accordingly, the combination of LXR and RARalpha agonists at concentrations found in human atherosclerotic plaques markedly enhances the capabilities of macrophages to engulf apoptotic cells in a TGM2-dependent manner., Conclusions: These results indicate an important role for LXRs in the control of phagocytosis through an RARalpha-TGM2-dependent mechanism. A combination of LXR/RARalpha agonists that may operate in atherosclerosis could also constitute a promising strategy to improve the clearance of apoptotic cells by macrophages in other pathological situations.

Ovarian hyperstimulation syndrome is a frequent complication occurring during in vitro fertilization cycles. It is characterized by a massive ovarian enlargement associated with an accumulation of extra vascular fluid. Here we show that liver X receptor (LXR)-alpha and LXR-beta deficient mice present many clinical and biological signs of ovarian hyperstimulation syndrome: ovarian enlargement, hemorrhagic corpora lutea, increased ovarian vascular permeability, and elevated estradiol. Ovulation stimulation resulted in excessive ovarian response to exogenous gonadotropins because follicle number and estradiol production were higher in transgenic mice. LXR deficiency also leads to perturbations in general inflammatory status, associated with ovarian il-6 deregulation. Upon treatment with the synthetic LXR agonist T09101317, serum estradiol and expression of star and cyp11a1 genes were markedly increased in wild-type mice, showing that LXRs are key regulators of ovarian steroidogenesis. These results suggest that LXRs control the ovulation by regulating endocrine and vascular processes.

Western diet is characterized by a hypercaloric and hyperlipidic intake, enriched in saturated fats, that is associated with the increased occurrence of metabolic diseases. To cope with this overload of dietary lipids, the intestine, which delivers dietary lipids to the body, has to adapt its capacity in lipid absorption and lipoprotein synthesis. We have studied the early effects of a high-fat diet (HFD) on intestinal lipid metabolism in mice. After 7 days of HFD, mice displayed normal fasting triglyceridemia but postprandial hypertriglyceridemia. HFD induced a decreased number of secreted chylomicrons with increased associated triglycerides. Secretion of larger chylomicrons was correlated with increased intestinal microsomal triglyceride transfer protein (MTP) content and activity. Seven days of HFD induced a repression of genes involved in fatty acid synthesis (FAS, ACC) and an increased expression of genes involved in lipoprotein assembly (apoB, MTP, and apoA-IV), suggesting a coordinated control of intestinal lipid metabolism to manage a high-fat loading. Of note, the mature form of the transcription factor SREBP-1c was increased and translocated to the nucleus, suggesting that it could be involved in the coordinated control of gene transcription. Activation of SREBP-1c was partly independent of LXR. Moreover, HFD induced hepatic insulin resistance whereas intestine remained insulin sensitive. Altogether, these results demonstrate that a short-term HFD is sufficient to impact intestinal lipid metabolism, which might participate in the development of dyslipidemia and metabolic diseases.

Oxysterol nuclear receptors liver x receptors (LXRalpha and LXRbeta) regulate lipid homeostasis when cells have to face high amounts of cholesterol and/or fatty acids. Male mice invalidated for both lxr (LXR-/-) are infertile by 5 months of age, and become sterile by the age of 9 months. The epididymis was previously shown to be affected by the gene invalidation, a phenotype specifically located in the two proximal segments of this organ. We demonstrate here that cholesteryl esters are accumulated in a specific cell type of the epididymal epithelium, the apical cells, in these two first segments, in LXR-/- male mice. These accumulations are correlated to a decrease in the amount of a specific membrane cholesterol transporter, ATP-binding cassette A1 (ABCA1) in the caput epididymidis of LXR-/- mice. This decrease is due to a transcriptional down-regulation, and we further demonstrate that ABCA1, in the two first segments of the caput epididymidis, is located in the apical cells, and that its accumulation is lost in these cells for LXR-/- male mice as soon as 4 months of age. These data bring new elements in the cholesterol trafficking pathways in the epididymis, and will help a better understanding of the molecular mechanisms occurring in this organ in relation to the sperm cells maturation process.

Liver X receptor-alpha (LXRalpha) and LXRbeta are ligand-activated transcription factors belonging to the nuclear receptor superfamily. They have been identified as key players in cholesterol homeostasis and lipid and glucose metabolism as well as immune and inflammatory responses. In the small intestine, LXRs have been shown not only to regulate cholesterol absorption and excretion but also to promote high-density lipoprotein biogenesis via the ATP-binding cassette A1 signaling pathway. Here, using gene expression assays, we identified PPARalpha as an intestine-specific LXR target gene. Chronic administration of LXR synthetic agonists led to a significant increase of PPARalpha mRNA levels in the small intestine but not in the liver. In addition, this specific PPARalpha gene up-regulation occurred in the duodenum, jejunum, and ileum in a dose-dependent manner and translated at the protein level as demonstrated by Western blot analysis. Furthermore, PPARalpha gene induction was completely abolished in LXR-deficient mice. Finally, the physiological relevance of LXR-mediated PPARalpha up-regulation in the small intestine was assessed in PPARalpha-deficient mice. Administration of a synthetic LXR agonist to wild-type mice led to the induction of several PPARalpha target genes including PDK4 and CPT1. Those effects were completely abolished in PPARalpha-deficient mice, demonstrating the biological relevance of this LXR-PPARalpha transcriptional cascade. Taken together, these results demonstrate that PPARalpha is an intestine-specific LXR target gene and suggest the existence of a transcriptional cross talk between those members of the nuclear receptor superfamily.

Human implantation involves invasion of the uterine wall and remodeling of uterine arteries by extravillous cytotrophoblasts. Defects in these early steps of placental development lead to poor placentation and are often associated with preeclampsia, a frequent complication of human pregnancy. One of the complex mechanisms controlling trophoblast invasion involves the activation of the liver X receptor beta (or NR1H2, more commonly known as LXRbeta) by oxysterols known as potent LXR activators. This activation of LXRbeta leads to a decrease of trophoblast invasion. The identification of new target genes of LXR in the placenta could aid in the understanding of their physiological roles in trophoblast invasion. In the present study, we show that the endoglin (ENG) gene is a direct target of the liver X receptor alpha (NR1H3, also known as LXRalpha). ENG, whose gene is highly expressed in syncytiotrophoblasts, is part of the transforming growth factor (TGF) receptor complex that binds several members of the TGFbeta superfamily. In the human placenta, ENG has been shown to be involved in the inhibition of trophoblast invasion. Treatment of human choriocarcinoma JAR cells with T0901317, a synthetic LXR-selective agonist, leads to a significant increase in ENG mRNA and protein levels. Using transfection and electrophoretic mobility shift assays, we demonstrate that LXR (as a heterodimer with the retinoid X receptor) is able to bind the ENG promoter on an LXR response element and mediates the activation of ENG gene expression by LXRalpha in JAR cells. This study suggests a novel mechanism by which LXR may regulate trophoblast invasion in pathological pregnancy such as preeclampsia.

The transcription factor carbohydrate-responsive element-binding protein (ChREBP) has emerged as a central regulator of lipid synthesis in liver because it is required for glucose-induced expression of the glycolytic enzyme liver-pyruvate kinase (L-PK) and acts in synergy with SREBP to induce lipogenic genes such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). Liver X receptors (LXRs) are also important regulators of the lipogenic pathway, and the recent finding that ChREBP is a direct target of LXRs and that glucose itself can bind and activate LXRs prompted us to study the role of LXRs in the induction of glucose-regulated genes in liver. Using an LXR agonist in wild-type mice, we found that LXR stimulation did not promote ChREBP phosphorylation or nuclear localization in the absence of an increased intrahepatic glucose flux. Furthermore, the induction of ChREBP, L-PK, and ACC by glucose or high-carbohydrate diet was similar in LXRalpha/beta knockout compared with wild-type mice, suggesting that the activation of these genes by glucose occurs by an LXR-independent mechanism. We used fluorescence resonance energy transfer analysis to demonstrate that glucose failed to promote the interaction of LXRalpha/beta with specific cofactors. Finally, siRNA silencing of ChREBP in LXRalpha/beta knockout hepatocytes abrogated glucose-induced expression of L-PK and ACC, further demonstrating the central role of ChREBP in glucose signaling. Taken together, our results demonstrate that glucose is required for ChREBP functional activity and that LXRs are not necessary for the induction of glucose-regulated genes in liver.

Aim: To investigate the roles of liver X receptors (LXR) in the lipid composition and gene expression regulation in the murine caput epididymidis. LXR are nuclear receptors for oxysterols, molecules derived from cholesterol metabolism that are present in mammals as two isoforms: LXRalpha, which is more specifically expressed in lipid-metabolising tissues, such as liver, adipose and steroidogenic tissues, and macrophages, whereas LXRbeta is ubiquitous. Their importance in reproductive physiology has been sustained by the fact that male mice in which the function of both LXR has been disrupted have fertility disturbances starting at the age of 5 months, leading to complete sterility by the age of 9 months. These defects are associated with epididymal epithelial degeneration in caput segments one and two, and with a sperm midpiece fragility, leading to the presence of isolated sperm heads and flagella when luminal contents are recovered from the cauda epididymidis., Methods: The lipid composition of the caput epididymidis of wild-type and LXR-deficient mice was assessed using oil red O staining on tissue cryosections and lipid extraction followed by high performance liquid chromatography or gas chromatography. Gene expression was checked by quantitative real time polymerase chain reaction., Results: Using LXR-deficient mice, we showed an alteration of the lipid composition of the caput epididymidis as well as a significantly decreased expression of the genes encoding SREBP1c, SCD1 and SCD2, involved in fatty acid metabolism., Conclusion: Altogether, these results show that LXR are important regulators of epididymal function, and play a critical role in the lipid maturation processes occurring during sperm epididymal maturation.

Oxysterol nuclear receptors liver X receptor (LXR)alpha and LXRbeta are known to regulate lipid homeostasis in cells exposed to high amounts of cholesterol and/or fatty acids. In order to elucidate the specific and redundant roles of the LXRs in the testis, we explored the reproductive phenotypes of mice deficient of LXRalpha, LXRbeta, and both, of which only the lxralpha;beta-/- mice are infertile by 5 months of age. We demonstrate that LXRalpha-deficient mice had lower levels of testicular testosterone that correlated with a higher apoptotic rate of the germ cells. LXRbeta-deficient mice showed increased lipid accumulation in the Sertoli cells and a lower proliferation rate of the germ cells. In lxralpha;beta-/- mice, fatty acid metabolism was affected through a decrease of srebp1c and increase in scd1 mRNA expression. The retinoid acid signaling pathway was also altered in lxralpha;beta-/- mice, with a higher accumulation of all-trans retinoid receptor alpha, all-trans retinoid receptor beta, and retinoic aldehyde dehydrogenase-2 mRNA. Combination of these alterations might explain the deleterious phenotype of infertility observed only in lxralpha;beta-/- mice, even though lipid homeostasis seemed to be first altered. Wild-type mice treated with a specific LXR agonist showed an increase of testosterone production involving both LXR isoforms. Altogether, these data identify new roles of each LXR, collaborating to maintain both integrity and functions of the testis.

The small heterodimer partner (SHP) is an atypical nuclear receptor known mainly for its role in bile acid homeostasis in the enterohepatic tract. We explore here the role of SHP in the testis. SHP is expressed in the interstitial compartment of the adult testes, which contain the Leydig cells. SHP there inhibits the expression of steroidogenic genes, on the one hand by inhibiting the expression of the nuclear receptors steroidogenic factor-1 and liver receptor homolog-1 (lrh-1), and on the other hand by directly repressing the transcriptional activity of LRH-1. Consequently, in SHP knockout mice, testicular testosterone synthesis is increased independently of the hypothalamus-pituitary axis. Independent of its action on androgen synthesis, SHP also determines the timing of germ cell differentiation by controlling testicular retinoic acid metabolism. Through the inhibition of the transcriptional activity of retinoic acid receptors, SHP controls the expression of stimulated by retinoic acid gene 8 (stra8) - a gene that is indispensable for germ cell meiosis and differentiation. Together, our data demonstrate new roles for SHP in testicular androgen and retinoic acid metabolism, making SHP a testicular gatekeeper of the timing of male sexual maturation.

Vitamin D is well known as a hormone involved in mineral metabolism and bone growth. Conversion into the active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) from the precursor is effected by cytochrome P450 enzymes in the liver (CYP27A1 and CYP2R1) and the kidney (CYP27B1). CYP27A1 has been shown to be transcriptionally regulated by nuclear receptors (PPARalpha, gamma, HNF-4alpha and SHP) which are ligand-dependent transcription factors. CYP27B1 is tightly regulated by the plasma levels of calcium, phosphate, parathyroid hormone (PTH) and 1,25(OH)2D3 itself. In vitamin D target organs, inactivation of vitamin D is attributed to CYP24A1 which is transcriptionally induced by 1,25(OH)2D3 whose action is mediated by binding to its cognate nuclear receptor, the vitamin D receptor (VDR). Diseases associated to Vitamin D deficiency (rickets in children, and osteomalacia or osteoporosis in adults) and autosomal recessive forms of inherited rickets illustrate the key role of vitamin D in calcium homeostasis and bone metabolism. Recently, discovery of 1,25(OH)2D3 new biological actions that include antiproliferative, prodifferentiating effect on many cell types and immunoregulatory properties creates a growing interest for this vitamin. In this way, a best understanding of various actors implicated in vitamin D metabolism and its regulation is of a major importance to optimise the use of vitamin D in disease prevention.

Cholesterol is the obligate precursor to adrenal steroids but is cytotoxic at high concentrations. Here, we show the role of the liver X receptors (LXRalpha and LXRbeta) in preventing accumulation of free cholesterol in mouse adrenal glands by controlling expression of genes involved in all aspects of cholesterol utilization, including the steroidogenic acute regulatory protein, StAR, a novel LXR target. Under chronic dietary stress, adrenal glands from Lxralphabeta-/- mice accumulated free cholesterol. In contrast, wild-type animals maintained cholesterol homeostasis through basal expression of genes involved in cholesterol efflux and storage (ABC transporter A1 [ABCA1], apoE, SREBP-1c) while preventing steroidogenic gene (StAR) expression. Upon treatment with an LXR agonist that mimics activation by oxysterols, expression of these target genes was increased. Basally, Lxralphabeta-/- mice exhibited a marked decrease in ABCA1 and a derepression of StAR expression, causing a net decrease in cholesterol efflux and an increase in steroidogenesis. These changes occurred under conditions that prevented the acute stress response and resulted in a phenotype more specific to the loss of LXRalpha, including hypercorticosteronemia, cholesterol ester accumulation, and adrenomegaly. These results imply LXRalpha provides a safety valve to limit free cholesterol levels as a basal protective mechanism in the adrenal gland, where cholesterol is under constant flux.

In this study we looked at the epididymides and spermatozoa of mice knocked-out for nuclear oxysterol receptors (LXR). We have shown that LXR-deficient mice exhibited upon ageing a severe disruption of their caput epididymides associated with abnormal accumulation of neutral lipids. The epididymis defaults were correlated with sperm head fragility and infertility. In agreement with the observed caput defect in transgenic animals in which both LXRalpha and LXRbeta isoforms were disrupted, we have shown here that both receptors are expressed in caput and cauda epididymides regions. LXRbeta was predominantly expressed throughout the mouse epididymis while the expression of LXRalpha was weaker. In addition, the expression of selected genes that can be considered as markers of adult epididymis function was monitored via Northern blots in the different single and double LXR-deficient backgrounds. Altogether, the data presented here suggest that LXR receptors are important actors in epididymis function.

Liver X receptors (LXRs) regulate the expression of a number of genes involved in cholesterol and lipid metabolism after activation by their cognate oxysterol ligands. AKR1-B7 (aldo-keto reductase 1-B7) is expressed in LXR target tissues such as intestine, and because of its known role in detoxifying lipid peroxides, we investigated whether the AKR1-B7 detoxification pathway was regulated by LXRs. Here we show that synthetic LXR agonists increase the accumulation of AKR1-B7 mRNA and protein levels in mouse intestine in wild-type but not lxr(-/-) mice. Regulation of akr1b7 by retinoic X receptor/LXR heterodimers is dependent on three response elements in the proximal murine akr1b7 promoter. Two of these cis-acting elements are specific for regulation by the LXRalpha isoform. In addition, in duodenum of wild-type mice fed a synthetic LXR agonist, we observed an LXR-dependent decrease in lipid peroxidation. Our results demonstrate that akr1b7 is a direct target of LXRs throughout the small intestine, and that LXR activation plays a protective role by decreasing the deleterious effects of lipid peroxides in duodenum. Taken together, these data suggest a new role for LXRs in lipid detoxification.

Liver X receptor-alpha and -beta are members of the nuclear hormone receptor superfamily that heterodimerize with retinoid X receptor and are activated by oxysterols. In recent studies we found that treatment of cultured human keratinocytes with oxysterolstimulated differentiation, as demonstrated by increased expression of involucrin and transglutaminase, and inhibited proliferation. The aims of this study were to determine: (i) whether oxysterols applied topically to the skin of mice induce differentiation in normal epidermis; (ii) whether this effect is mediated via liver X receptor-alpha and/or liver X receptor-beta; and (iii) whether oxysterols normalize epidermal morphology in an animal model of epidermal hyperplasia. Topical treatment of normal hairless mice with 22(R)-hydroxycholesterol or 24(S),25-epoxycholesterol resulted in a decrease in epidermal thickness and a decrease in keratinocyte proliferation assayed by proliferating cell nuclear antigen staining. Moreover, oxysterol treatment increased the levels of involucrin, loricrin, and profilaggrin protein and mRNA in the epidermis, indicating that oxysterols stimulate epidermal differentiation. Additionally, topical oxysterol pretreatment improved permeability barrier homeostasis. Whereas liver X receptor-alpha-/- mice revealed no alterations in epidermal differentiation, the epidermis was thinner in liver X receptor-beta-/- mice than in wild-type mice, with a reduced number of proliferating cell nuclear antigen positive cells and a modest reduction in the expression of differentiation markers. Topical oxysterol treatment induced differentiation in liver X receptor-alpha-/- mice whereas in liver X receptor-beta-/- mice there was no increase in the expression of differentiation markers. Whereas both liver X receptor-alpha and liver X receptor-beta are expressed in cultured human keratinocytes and in fetal rat skin, only liver X receptor-beta was observed on northern blotting in adult mouse epidermis. Finally, treatment of hyperproliferative epidermis with oxysterols restored epidermal homeostasis. These studies demonstrate that epidermal differentiation is regulated by liver X receptor-beta and that oxysterols, acting via liver X receptor-beta, can induce differentiation and inhibit proliferation in vivo. The ability of oxysterols to reverse epidermal hyperplasia suggests that these agents could be beneficial for the treatment of skin disorders associated with hyperproliferation and/or altered differentiation.

A common feature of many metabolic pathways is their control by retinoid X receptor (RXR) heterodimers. Dysregulation of such metabolic pathways can lead to the development of atherosclerosis, a disease influenced by both systemic and local factors. Here we analyzed the effects of activation of RXR and some of its heterodimers in apolipoprotein E -/- mice, a well established animal model of atherosclerosis. An RXR agonist drastically reduced the development of atherosclerosis. In addition, a ligand for the peroxisome proliferator-activated receptor (PPAR)gamma and a dual agonist of both PPARalpha and PPARgamma had moderate inhibitory effects. Both RXR and liver X receptor (LXR) agonists induced ATP-binding cassette protein 1 (ABC-1) expression and stimulated ABC-1-mediated cholesterol efflux from macrophages from wild-type, but not from LXRalpha and beta double -/-, mice. Hence, activation of ABC-1-mediated cholesterol efflux by the RXR/LXR heterodimer might contribute to the beneficial effects of rexinoids on atherosclerosis and warrant further evaluation of RXR/LXR agonists in prevention and treatment of atherosclerosis.

The liver X receptors (LXRs) are members of the nuclear hormone receptor superfamily that are bound and activated by oxysterols. These receptors serve as sterol sensors to regulate the transcription of gene products that control intracellular cholesterol homeostasis through catabolism and transport. In this report, we describe a novel LXR target, the sterol regulatory element-binding protein-1c gene (SREBP-1c), which encodes a membrane-bound transcription factor of the basic helix-loop-helix-leucine zipper family. SREBP-1c expression was markedly increased in mouse tissues in an LXR-dependent manner by dietary cholesterol and synthetic agonists for both LXR and its heterodimer partner, the retinoid X receptor (RXR). Expression of the related gene products, SREBP-1a and SREBP-2, were not increased. Analysis of the mouse SREBP-1c gene promoter revealed an RXR/LXR DNA-binding site that is essential for this regulation. The transcriptional increase in SREBP-1c mRNA by RXR/LXR was accompanied by a similar increase in the level of the nuclear, active form of the SREBP-1c protein and an increase in fatty acid synthesis. Because this active form of SREBP-1c controls the transcription of genes involved in fatty acid biosynthesis, our results reveal a unique regulatory interplay between cholesterol and fatty acid metabolism.

To identify genes that are transcriptionally activated when human macrophages accumulate excess lipids, we employed the mRNA differential display technique using RNA isolated from human monocyte-macrophages incubated in the absence or presence of acetylated low density lipoprotein and sterols (cholesterol and 25-hydroxycholesterol). These studies identified a mRNA whose levels were highly induced in lipid-loaded macrophages. The mRNA encoded the human White protein, a member of the ATP-binding cassette (ABC) transporter superfamily of proteins. The mRNA levels of ABC8, the murine homolog of the human white gene, were also induced when a murine macrophage cell line, RAW264.7, was incubated with acetylated low density lipoprotein and sterols. Additional studies demonstrated that white/ABC8 mRNA levels were induced by specific oxysterols that included 25-, 20(S)-, and 22(R)-hydroxycholesterol, and by a retinoid X receptor-specific ligand. Furthermore, the oxysterol-mediated induction of ABC8 expression in mouse peritoneal macrophages was dependent on the presence of the nuclear oxysterol receptors, liver X receptors (LXRs). Macrophages derived from mice lacking both LXRalpha and LXRbeta failed to up-regulate the expression of ABC8 following incubation with 22(R)-hydroxycholesterol. Oxysterol-dependent induction of white/ABC8 mRNA was blocked by actinomycin D but not by cycloheximide treatment of cells. We conclude that the white and ABC8 genes are primary response genes that are transcriptionally activated by specific oxysterols and that this induction is mediated by the LXR subfamily of nuclear hormone receptors. These data strongly support the hypothesis that white/ABC8 has a role in cellular sterol homeostasis.

We investigated the interferences of the normal or mutated androgen receptor with the activator protein-1 (AP-1) by assessing their effects on transcriptional activity in CV-1 cells. A luciferase reporter gene was constructed downstream from either a promoter for the mouse vas deferens protein, or a trimerized 12-O-tetradecanoyl phorbol-13-acetate-response element site whose transcriptions are activated by androgen and 12-O-tetradecanoyl phorbol-13-acetate, a potent AP-1 activator. The blockade of dephosphorylation by protein phosphatases identifies the protein phosphatases that modulate the AP-1/androgen receptor cross-talk. Using engineered or naturally occurring androgen receptor mutants that are responsible for complete or partial androgen insensitivity syndromes, we defined the subregions involved in the cross-talk of the androgen receptor with the AP-1 factors. First, it appears that the 188 first amino acids of the N-terminal domain of the androgen receptor are necessary to obtain a full transrepression. Second, a functional and intact ligand binding domain is critical for the modulation of androgen/AP-1 pathway interactions. Third, normal DNA binding capacity of the androgen receptor is not required. Two mutants at positions 568 and 581 of the DNA binding domain demonstrate that the transactivation and transrepression functions of the androgen receptor can be dissociated. Collectively, these data indicate that several segments of the androgen receptor are involved in cross-talk with the AP-1 pathway. Mutations within the DNA binding domain of the androgen receptor highly impair these interferences.

We demonstrate that mice lacking the oxysterol receptor, LXR alpha, lose their ability to respond normally to dietary cholesterol and are unable to tolerate any amount of cholesterol in excess of that which they synthesize de novo. When fed diets containing cholesterol, LXR alpha (-/-) mice fail to induce transcription of the gene encoding cholesterol 7alpha-hydroxylase (Cyp7a), the rate-limiting enzyme in bile acid synthesis. This defect is associated with a rapid accumulation of large amounts of cholesterol in the liver that eventually leads to impaired hepatic function. The regulation of several other crucial lipid metabolizing genes is also altered in LXR alpha (-/-) mice. These results demonstrate the existence of a physiologically significant feed-forward regulatory pathway for sterol metabolism and establish the role of LXR alpha as the major sensor of dietary cholesterol.

In the androgen receptor of a patient with androgen insensitivity, the alanine residue at position 564 in the first zinc cluster of the DNA-binding domain was substituted by aspartic acid. In other members of the steroid receptor family, either valine or alanine is present at the corresponding position, suggesting the importance of a neutral amino acid residue at this site. The mutant receptor was transcriptionally inactive, which corresponded to the absence of specific DNA binding in gel retardation assays, and its inactivity in a promoter interference assay. Two other receptor mutants with a mutation at this same position were created to study the role of position 564 in the human androgen receptor on DNA binding in more detail. Introduction of asparagine at position 564 resulted in transcription activation of a mouse mammary tumor virus promoter, although at a lower level compared with the wild-type receptor. Transcription activation of an (ARE)2-TATA promoter was low, and binding to different hormone response elements could not be visualized. The receptor with a leucine residue at position 564 was as active as the wild-type receptor on a mouse mammary tumor virus promoter and an (ARE)2-TATA promoter, but interacted differentially with several hormone response elements in a gel retardation assay. The results of the transcription activation and DNA binding studies could partially be predicted from three-dimensional modeling data. The phenotype of the patient was explained by the negative charge, introduced at position 564.