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STUDY QUESTION Are low or high plasma mannose-binding lectin (p-MBL) levels associated with recurrent pregnancy loss (RPL) and the reproductive and perinatal outcomes before and after RPL? SUMMARY ANSWER The prevalence of low p-MBL levels was significantly higher in RPL patients, while high levels were significantly less prevalent. No association was found between p-MBL level and reproductive and perinatal outcomes before and after RPL. WHAT IS KNOWN ALREADY Mannose-binding lectin (MBL) is an important component in the innate immune system. Low p-MBL levels have been associated with RPL, while the correlation with high levels has been poorly studied. Adverse perinatal outcomes are generally more frequent among RPL patients, but reports concerning the association between maternal p-MBL levels and perinatal outcomes, including birth weight (BW) and gestational age (GA), are conflicting. STUDY DESIGN, SIZE, DURATION This study was a combined cross-sectional and cohort study of 267 RPL patients admitted to the RPL Center of Western Denmark between January 2016 and March 2020. RPL patients were followed until birth of a liveborn child or until end of follow-up, March 2021. A sample of 185 healthy female blood donors of reproductive age was used as a MBL reference group. PARTICIPANTS/MATERIALS, SETTING, METHODS All RPL patients had ≥3 consecutive pregnancy losses, a regular menstrual cycle and no known significant chromosomal or uterine malformations. At the first consultation, routine blood samples including p-MBL measurement and detailed obstetrical and perinatal information were collected. p-MBL levels in RPL patients were compared to the MBL reference group. A logistic regression analysis adjusted for relevant confounders assessed the association between low p-MBL levels and an unsuccessful reproductive outcome in RPL patients in first pregnancy after admission. Perinatal outcomes before and after RPL were compared between RPL subgroups according to low (≤500 µg/l), intermediate (501–3000 µg/l) and high (>3000 µg/l) p-MBL levels. MAIN RESULTS AND THE ROLE OF CHANCE Significantly more RPL patients had low p-MBL levels (prevalence proportion ratio (PPR): 1.79, 95% CI: 1.34–2.38) and fewer had high p-MBL levels (PPR: 0.56, 95% CI: 0.40–0.79) compared to the reference group, while the prevalence of intermediate p-MBL level was not different between the groups (PPR: 0.86, 95% CI: 0.69–1.08). In the prospective study, low p-MBL level was not a significant risk factor for a pregnancy loss in the first pregnancy after admission after adjustment for age, BMI and smoking. Neither before nor after the RPL diagnosis were maternal p-MBL levels significantly associated with BW or GA. LIMITATIONS, REASONS FOR CAUTION Only 161 (60.3%) patients had given birth after RPL during the follow-up period, which limited the possibility to detect clear associations between p-MBL levels and perinatal outcomes after RPL. WIDER IMPLICATIONS OF THE FINDINGS In agreement with several previous studies, low p-MBL levels are strongly associated with RPL, while this study for the first time documents that high levels may play a protective role, which suggests a causal relationship. We suggest that larger prospective studies evaluate the association between p-MBL levels and RPL prognosis. STUDY FUNDING/COMPETING INTEREST(S) No external funding was received. We acknowledge the Department of Obstetrics and Gynaecology at Aalborg University Hospital for financial support. U.S.K. has reported personal fees from Merck, consulting fees from IBSA Nordic, and a grant from Gedeon Richter, Merck and IBSA Nordic outside of the submitted work. TRIAL REGISTRATION NUMBER ID from clinicaltrials.gov is NCT04017754.

Until three decades, ago 3,5-diiodothyronine (3,5-T2) and 3,3'-diiodothyronine (3,3'-T2) were considered products of thyroid hormone catabolism without biological activity. Some metabolic effects have been described in rodents, but the physiological relevance in humans and the mechanisms of action are unknown. Aim of this work was to investigate the role and the mechanisms of action of 3,5-T2 and 3,3'-T2 in the regulation of metabolic homeostasis in human liver. We used primary human hepatocytes freshly isolated from donors and grown on Matrigel as the golden standard in vitro model to study human hepatic metabolism. Results show that diiodothyronines in the range of plasma physiological concentrations reduced hepatic lipid accumulation, by modulating the activity of the mTORC1/Raptor complex through an AMPK-mediated mechanism, and stimulated the mTORC2/Rictor complex-activated pathway, leading to the down regulation of the expression of key gluconeogenic genes. Hence, we propose that diiodothyronines act as key regulators of hepatic metabolic homeostasis in humans.

Introduction Liver X receptors (LXRs) are nuclear receptors that function as cholesterol sensors and regulate cholesterol homeostasis. High cholesterol has been recognized as a risk factor in asthma; however, the mechanism of this linkage is not known. Methods To explore the importance of cholesterol homeostasis for asthma, we investigated the contribution of LXR activity in an ovalbumin- and a house dust mite-driven eosinophilic asthma mouse model. Results In both models, airway inflammation, airway hyper-reactivity, and goblet cell hyperplasia were reduced in mice deficient for both LXRα and LXRβ isoforms (LXRα−/−β−/−) as compared to wild-type mice. Inversely, treatment with the LXR agonist GW3965 showed increased eosinophilic airway inflammation. LXR activity contributed to airway inflammation through promotion of type 2 cytokine production as LXRα−/−β−/− mice showed strongly reduced protein levels of IL-5 and IL-13 in the lungs as well as reduced expression of these cytokines by CD4+ lung cells and lung-draining lymph node cells. In line herewith, LXR activation resulted in increased type 2 cytokine production by the lung-draining lymph node cells. Conclusions In conclusion, our study demonstrates that the cholesterol regulator LXR acts as a positive regulator of eosinophilic asthma in mice, contributing to airway inflammation through regulation of type 2 cytokine production.

Depletion of the cholesterol esterifying enzyme acyl-Coenzyme A: cholesterol acyltransferase 2 (ACAT2, encoded by Soat2) protects mice from atherosclerosis, diet-induced hypercholesterolemia, and hepatic steatosis when fed high-cholesterol diet. The glucose transporter 2 (GLUT2) represents the main gate of glucose uptake by the liver. Lipid synthesis from glucose (de novo lipogenesis; DNL) plays a pivotal role in the development of hepatic steatosis. Inhibition of DNL is a successful approach to reverse hepatic steatosis, as shown by different studies in mice and humans. Here we aimed to investigate whether depletion of Soat2 per se can reduce hepatic steatosis, also in the presence of very low levels of cholesterol in the diet, and the underlying mechanisms. Female Soat2-/- and wild type mice were either fed high-fat or high-carbohydrate diet and both contained

Summary Non-alcoholic fatty liver disease (NAFLD) is a very common indication for liver transplantation. How fat-rich diets promote progression from fatty liver to more damaging inflammatory and fibrotic stages is poorly understood. Here, we show that disrupting phosphorylation at Ser196 (S196A) in the liver X receptor alpha (LXRα, NR1H3) retards NAFLD progression in mice on a high-fat-high-cholesterol diet. Mechanistically, this is explained by key histone acetylation (H3K27) and transcriptional changes in pro-fibrotic and pro-inflammatory genes. Furthermore, S196A-LXRα expression reveals the regulation of novel diet-specific LXRα-responsive genes, including the induction of Ces1f, implicated in the breakdown of hepatic lipids. This involves induced H3K27 acetylation and altered LXR and TBLR1 cofactor occupancy at the Ces1f gene in S196A fatty livers. Overall, impaired Ser196-LXRα phosphorylation acts as a novel nutritional molecular sensor that profoundly alters the hepatic H3K27 acetylome and transcriptome during NAFLD progression placing LXRα phosphorylation as an alternative anti-inflammatory or anti-fibrotic therapeutic target., Graphical Abstract, Highlights • LXRαS196A induces liver steatosis and prevents cholesterol accumulation • LXRαS196A reduces progression to hepatic inflammation and fibrosis • LXRαS196A modulates hepatic chromatin acetylation • LXRαS196A reveals unique dual LXRα phosphorylation and diet-responsive genes, Progression to inflammatory and fibrotic steatohepatitis is poorly understood. Becares et al. reveal that disrupting LXRα phosphorylation attenuates these processes by promoting a unique diet-induced transcriptome that prevents cholesterol accumulation and reduces hepatic inflammation and fibrosis. Mechanistically, phospho-deficient LXRα promotes chromatin modifications and alters protein-protein interactions in differentially expressed genes.

Liver X receptor (LXR)α and LXRβ play key roles in hepatic de novo lipogenesis through their regulation of lipogenic genes, including sterol regulatory element-binding protein (SREBP)-1c and carbohydrate responsive element-binding protein (ChREBP). LXRs activate lipogenic gene transcription in response to feeding, which is believed to be mediated by insulin. We have previously shown that LXRs are targets for glucose-hexosamine-derived O-linked β-N-acetylglucosamine (O-GlcNAc) modification enhancing their ability to regulate SREBP-1c promoter activity in vitro. To elucidate insulin-independent effects of feeding on LXR-mediated lipogenic gene expression in vivo, we subjected control and streptozotocin-treated LXRα/β(+/+) and LXRα/β(-/-) mice to a fasting-refeeding regime. We show that under hyperglycemic and hypoinsulinemic conditions, LXRs maintain their ability to upregulate the expression of glycolytic and lipogenic enzymes, including glucokinase (GK), SREBP-1c, ChREBPα, and the newly identified shorter isoform ChREBPβ. Furthermore, glucose-dependent increases in LXR/retinoid X receptor-regulated luciferase activity driven by the ChREBPα promoter was mediated, at least in part, by O-GlcNAc transferase (OGT) signaling in Huh7 cells. Moreover, we show that LXR and OGT interact and colocalize in the nucleus and that loss of LXRs profoundly reduced nuclear O-GlcNAc signaling and ChREBPα promoter binding activity in vivo. In summary, our study provides evidence that LXRs act as nutrient and glucose metabolic sensors upstream of ChREBP by modulating GK expression, nuclear O-GlcNAc signaling, and ChREBP expression and activity.

Understanding the transition from fatty liver or steatosis to more advanced inflammatory and fibrotic stages of non-alcoholic fatty liver disease (steatohepatitis), is key to define strategies that alter or even reverse the progression of this pathology. The Liver X Receptor alpha (LXRα) controls hepatic lipid homeostasis and inflammation. Here we show that mice carrying a mutation that abolishes phosphorylation at Ser196 (S196A) in LXRα exhibit reduced hepatic inflammation and fibrosis when challenged with a high fat-high cholesterol diet, despite displaying enhanced hepatic lipid accumulation. This protective effect is associated with reduced cholesterol accumulation, a key promoter of lipid-mediated hepatic damage. Reduced steatohepatitis in S196A mice involves the reprogramming of the liver transcriptome by promoting diet-induced changes in the expression of genes involved in endoplasmic reticulum stress, extracellular matrix remodelling, inflammation and lipid metabolism. Unexpectedly, changes in LXRα phosphorylation uncover novel diet-specific target genes, whose regulation does not simply mirror ligand-induced LXR activation. These unique LXRα phosphorylation-sensitive, diet-responsive target genes are revealed by promoting LXR occupancy and cofactor recruitment in the context of a cholesterol-rich diet. Therefore, LXRα phosphorylation at Ser196 critically acts as a novel nutritional sensor that promotes a unique diet-induced transcriptome thereby modulating metabolic, inflammatory and fibrotic responses important in the transition to steatohepatitis.

Oxysterols are involved in maintaining cellular cholesterol levels. Recently, oxysterols have been demonstrated to modulate the function of immune cells and tumor growth. These effects can be dependent on the activation of the oxysterol-binding liver X receptors (LXRs) or, as recently demonstrated for T and B cells, DCs and neutrophils, can be independent of LXR activation. LXR-dependent oxysterol effects can be ascribed to the activation of LXRα, LXRβ or LXRαβ isoforms, which induces transcriptional activation or trans-repression of target genes. The prevalent activation of one isoform seems to be cell-, tissue-, or context-specific, as shown in some pathologic processes, i.e., infectious diseases, atherosclerosis, and autoimmunity. Oxysterol-LXR signaling has recently been shown to inhibit antitumor immune responses, as well as to modulate tumor cell growth. Here, we review the mechanisms that link oxysterols to tumor growth, and discuss possible networks at the basis of LXR-dependent and -independent oxysterol effects on immune cells and tumor development.

We investigated whether: (1) liver X receptor (LXR)-driven induction of high-density lipoprotein cholesterol (HDL-C) and other LXR-mediated effects on cholesterol metabolism depend on intestinal cholesterol absorption; and (2) combined treatment with the LXR agonist GW3965 and the cholesterol absorption inhibitor ezetimibe results in synergistic effects on cholesterol metabolism that could be beneficial for treatment of atherosclerosis. Mice were fed 0.2 % cholesterol and treated with GW3965+ezetimibe, GW3965 or ezetimibe. GW3965+ezetimibe treatment elevated serum HDL-C and Apolipoprotein (Apo) AI, effectively reduced the intestinal cholesterol absorption and increased the excretion of faecal neutral sterols. No changes in intestinal ATP-binding cassette (ABC) A1 or ABCG5 protein expression were observed, despite increased mRNA expression, while hepatic ABCA1 was slightly reduced. The combined treatment caused a pronounced down-regulation of intestinal Niemann-Pick C1-like 1 (NPC1L1) and reduced hepatic and intestinal cholesterol levels. GW3965 did not affect the intestinal cholesterol absorption, but increased serum HDL-C and ApoAI levels. GW3965 also increased Apoa1 mRNA levels in primary mouse hepatocytes and HEPA1-6 cells. Ezetimibe reduced the intestinal cholesterol absorption, ABCA1 and ABCG5, but did not affect the serum HDL-C or ApoAI levels. Thus, the LXR-driven induction of HDL-C and ApoAI was independent of the intestinal cholesterol absorption and increased expression of intestinal or hepatic ABCA1 was not required. Inhibited influx of cholesterol via NPC1L1 and/or low levels of intracellular cholesterol prevented post-transcriptional expression of intestinal ABCA1 and ABCG5, despite increased mRNA levels. Combined LXR activation and blocked intestinal cholesterol absorption induced effective faecal elimination of cholesterol.

The human major histocompatibility complex class II isotype HLA-DR is currently used as an activation marker for T cells. However, whether an endogenous protein expression or a molecular acquisition accounts for the presence of HLA-DR on T cells remains undetermined and still controversial. To further characterize this phenomenon, we compared several aspects of the presence of the HLA-DR protein to the presence of associated mRNA (HLA-DRB1), focusing on human T cells from peripheral blood of healthy individuals. Using a flow cytometric approach, we determined that the HLA-DR observed on CD4(+) T cells was almost exclusively cell surface-associated, while for autologous CD19(+) B cells, the protein could be located in the plasma membrane as well as in the cytoplasm. Moreover, negligible expression levels of HLA-DRB1 were found in CD4(+) T cells, using an HLA-DRB1 allele-specific qPCR assay. Finally, the presence of HLA-DR was not confined to activated CD4(+) and CD8(+) T cells, as evaluated by the co-expression of CD25. The functional role of the HLA-DR molecule on T cells remains enigmatic; however, this study presents evidence of fundamental differences for the presence of HLA-DR on T cells from HLA-DR in the context of antigen-presenting cells, which is a well-known phenomenon. Although an inducible endogenous protein expression cannot be excluded for the T cells, our findings suggest that a re-evaluation of the HLA-DR as a T cells activation marker is warranted.

Liver X receptors (LXRs) are nuclear receptors with well-known functions in cholesterol transport, fatty acid and glucose metabolism, and modulation of immune responses. Natural and synthetic ligands have been identified and are under development for the treatment of metabolic and inflammatory conditions and diseases. There is mounting evidence pointing to functional roles for LXRs in a variety of malignancies and the potential therapeutic efficacy of their ligands.This review summarizes the discovery and characterization of LXRs and their ligands, surveys their effects and mechanisms of action in cell-based and animal models of cancer, and proposes the future direction of basic and translational studies of LXRs and their ligands in cancer research and therapeutics.Targeting LXRs is a promising strategy for cancer treatment, particularly for those cancers which do not have effective treatment options. Key questions remain, however, regarding the specific mechanisms of action, effects on other target cells within the tumor microenvironment, and receptor status in patient populations. Moreover, LXR ligands optimized for disease-specific functions and cancer-related endpoints are currently not available. These issues represent both challenges and significant opportunities for future research and development efforts.

Elevated CRP and matrix metalloproteinase-9 associate with increased risk of cardiovascular events, possibly because these plasma proteins mark vulnerable atherosclerotic plaques. We tested the hypothesis that levels of C-reactive protein (CRP) and matrix metalloproteinase-9 associate with prognosis in patients with stable coronary heart disease.We measured baseline plasma CRP and matrix metalloproteinase-9 in 1090 patients with stable coronary heart disease and as the primary composite endpoint detected incident unstable angina, myocardial infarction and any death during 15 years of follow-up. CRP above versus below the median of 3.0 mg/L was associated with an increased cumulative incidence of unstable angina, myocardial infarction and any death combined (log-rank p0.0001). CRP above versus below the median had a corresponding hazard ratio of 1.5(95% CI, 1.3-1.8) after age adjustment, of 1.4(1.2-1.6) after multifactorial adjustment, and of 1.4(1.2-1.6) after multifactorial adjustment including degree of coronary disease. In contrast, matrix metalloproteinase-9 above versus below the median was not associated with risk of unstable angina, myocardial infarction and death.Elevated CRP, but not elevated matrix metalloproteinase-9, associates with increased risk of unstable angina, myocardial infarction and death in patients with stable coronary heart disease.

Liver X receptor (LXR)α regulates the genes involved in cholesterol, fatty acid and glucose metabolism. Soy protein (SP) consumption reduces the hepatic accumulation of cholesterol and triacylglycerol, and improves insulin sensitivity. However, it is not known whether these effects are mediated via LXRα. We therefore investigated whether the consumption of SP regulates metabolic changes in cholesterol metabolism and insulin sensitivity via LXRα. Wild-type (WT) and Lxrα −/− (Lxrα, also known as Nr1h3) mice were fed an SP diet with or without cholesterol for 28 days. The expression of LXRα target genes was measured in liver and intestine, as were hepatic lipid content and faecal bile acid concentration. Oral glucose and insulin tolerance tests were also performed. Hepatocytes were used to study the effect of isoflavones on LXR activity. The livers of WT and Lxrα −/− mice fed an SP high-cholesterol diet showed less steatosis than those fed casein. The SP diet increased the expression of the ATP-binding cassette (ABC) sub-family genes Abca1, Abcg5 and Abcg8 in the liver and intestine, as well as increasing total faecal bile acid excretion and insulin sensitivity in WT mice compared with mice fed a casein diet. However, these effects of SP were not observed in Lxrα −/− mice. The SP isoflavone, genistein, repressed the activation of LXRα target genes by T0901317, whereas it stimulated the activation of LXRβ target genes. The AMP-activated protein kinase inhibitor, compound C, had the opposite effects to those of genistein. Our results suggest that SP isoflavones stimulate the phosphorylation of LXRα or LXRβ, resulting in different biological effects for each LXR isoform.

The organ content of the mevalonate pathway lipids was investigated in liver-X-receptor (LXR) α, β and double knock-out mice. An extensive or moderate increase of total cholesterol in the double KO mice was found in all organs elicited by the increase of the esterified form. In LXRα and double KO mice, coenzyme Q (CoQ) was decreased in liver and increased in spleen, thymus and lung, while dolichol was increased in all organs investigated. This effect was confirmed using LXR- agonist GW 3965. Analysis of CoQ distribution in organelles showed that the modifications are present in all cellular compartments and that the increase of the lipid in mitochondria was the result of a net increase of CoQ without changing the number of mitochondria. It appears that LXR influences not only cellular cholesterol homeostasis but also the metabolism of CoQ and dolichol, in an indirect manner.

The oxysterol receptors LXRα and LXRβ are members of the nuclear receptor family and established transcriptional regulators of lipid metabolism with additional anti-inflammatory functions. Recent investigations have indicated an important role of LXRs in the control of proliferation. Here we further extend this knowledge to human colon cancer cells and proliferation in mouse colon. We show that activation of LXRs leads to a robust cell cycle arrest in colorectal adenocarcinoma cell lines. At the molecular level LXRs control expression of several cell cycle genes including Skp2, c-Myc, CDKs, cyclins, and p15. Furthermore, activation of LXRs causes hypo-phosphorylation of the retinoblastoma (Rb) tumor suppressor protein. Experiments performed in vivo show that the colon structure appears to be intact in LXR null mice. However, LXRαβ(-/-) mice show a significant increase of proliferation markers in colon compared to wild type mice and administration of the LXR specific agonist, GW3965 significantly reduced expression of proliferation in mouse colon. Taken together, these findings point toward a strong anti-proliferative effect of LXRs in colon revealing the potential of LXR ligands as possible anti cancer agents.

Hu X, Steffensen KR, Jiang Z-Y, Parini P, Gustafsson J-A, Gafvels M, Eggertsen G (Karolinska Institutet, Huddinge, Stockholm, Sweden; Karolinska Institutet, Huddinge, Stockholm, Sweden; Shanghai Institute of Digestive Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China; and University of Houston, Houston, TX, USA). LXRβ activation increases intestinal cholesterol absorption, leading to an atherogenic lipoprotein profile. J Intern Med 2012; 272: 452–464. Objectives. Liver X receptors (LXRs) are essential for the regulation of intestinal cholesterol absorption. Because two isoforms exist, LXRα and LXRβ, with overlapping but not identical functions, we investigated whether LXRα and LXRβ exert different effects on intestinal cholesterol absorption. Design. Wild-type (WT), LXRα−/− and LXRβ−/− mice were fed control diet, 0.2% cholesterol-enriched diet or 0.2% cholesterol-enriched diet plus the LXR agonist GW3965. Results. When fed a control diet, all three genotypes showed similar levels of cholesterol absorption. Of interest, a significant increase in cholesterol absorption was found in the LXRα−/− mice, but not in the WT or LXRβ−/− animals, when fed a diet enriched with 0.2% cholesterol or 0.2% cholesterol + GW3965. Reduced faecal neutral sterol excretion and a hydrophobic bile acid profile were also observed in LXRα−/− mice. Greater increases in the apolipoprotein (apo)B-containing lipoproteins in serum were seen in the LXRα−/− mice. A 0.2% cholesterol + GW3965 diet suppressed intestinal Npc1l1 protein expression to the same extent for all genotypes, while Abca1 and Abcg5 were elevated to the same degree. Conclusions. In the intestine, LXRα and LXRβ seem to exert similar effects on expression of cholesterol-transporting proteins such as Npc1l1. Selective activation of LXRβ may generate effects such as increased cholesterol absorption and elevated serum levels of apoB-containing lipoproteins, which seem to be counteracted by LXRα. Therefore, an intestinal LXRβ-specific pathway might exist in terms of cholesterol transportation in addition to the main pathway.

The liver X receptors (LXRs) are nuclear receptors that form permissive heterodimers with retinoid X receptor (RXR) and are important regulators of lipid metabolism in the liver. We have recently shown that RXR agonist-induced hypertriglyceridemia and hepatic steatosis in mice are dependent on LXRs and correlate with an LXR-dependent hepatic induction of lipogenic genes. To further investigate the roles of RXR and LXR in the regulation of hepatic gene expression, we have mapped the ligand-regulated genome-wide binding of these factors in mouse liver. We find that the RXR agonist bexarotene primarily increases the genomic binding of RXR, whereas the LXR agonist T0901317 greatly increases both LXR and RXR binding. Functional annotation of putative direct LXR target genes revealed a significant association with classical LXR-regulated pathways as well as peroxisome proliferator-activated receptor (PPAR) signaling pathways, and subsequent chromatin immunoprecipitation-sequencing (ChIP-seq) mapping of PPARα binding demonstrated binding of PPARα to 71 to 88% of the identified LXR-RXR binding sites. The combination of sequence analysis of shared binding regions and sequential ChIP on selected sites indicate that LXR-RXR and PPARα-RXR bind to degenerate response elements in a mutually exclusive manner. Together, our findings suggest extensive and unexpected cross talk between hepatic LXR and PPARα at the level of binding to shared genomic sites.

The acute phase response (APR) classically refers to the rapid reprogramming of gene expression and metabolism in response to inflammatory cytokine signaling. As components of the innate immune system, hepatocyte-derived acute phase proteins (APPs) play a central role in restoring tissue homeostasis. Recently, an intriguing 'metaflammatory' facet of the APR became evident with chronically elevated APP levels being connected to metabolic syndrome disorders. The causality of these connections is unclear but could relate to adverse metabolic and inflammatory disturbances, particularly those affecting lipoprotein properties, cholesterol metabolism and atherogenesis. Here we review these aspects with an emphasis on the emerging importance of lipid-sensing nuclear receptors (LXRs, LRH-1, PPARs), in conjunction with anti-inflammatory transrepression pathways, as physiological and pharmacological relevant modulators of the APR.

The liver X receptors (LXRs) play a key role in cholesterol and bile acid metabolism but are also important regulators of glucose metabolism. Recently, LXRs have been proposed as a glucose sensor affecting LXR-dependent gene expression. We challenged wild-type (WT) and LXRαβ−/−mice with a normal diet (ND) or a high-carbohydrate diet (HCD). Magnetic resonance imaging showed different fat distribution between WT and LXRαβ−/−mice. Surprisingly, gonadal (GL) adipocyte volume decreased on HCD compared with ND in WT mice, whereas it slightly increased in LXRαβ−/−mice. Interestingly, insulin-stimulated lipogenesis of isolated GL fat cells was reduced on HCD compared with ND in LXRαβ−/−mice, whereas no changes were observed in WT mice. Net de novo lipogenesis (DNL) calculated from V̇o2and V̇co2was significantly higher in LXRαβ−/−than in WT mice on HCD. Histology of HCD-fed livers showed hepatic steatosis in WT mice but not in LXRαβ−/−mice. Glucose tolerance was not different between groups, but insulin sensitivity was decreased by the HCD in WT but not in LXRαβ−/−mice. Finally, gene expression analysis of adipose tissue showed induced expression of genes involved in DNL in LXRαβ−/−mice compared with WT animals as opposed to the liver, where expression of DNL genes was repressed in LXRαβ−/−mice. We thus conclude that absence of LXRs stimulates DNL in adipose tissue, but suppresses DNL in the liver, demonstrating opposite roles of LXR in DNL regulation in these two tissues. These results show tissue-specific regulation of LXR activity, a crucial finding for drug development.

Liver X receptors (LXRs) regulate immune cell function and cholesterol metabolism, both factors that are critically involved in Alzheimer's disease (AD). To investigate the therapeutic potential of long-term LXR activation in amyloid-β (Aβ) peptide deposition in an AD model, 13-month-old, amyloid plaque-bearing APP23 mice were treated with the LXR agonist TO901317. Postmortem analysis demonstrated that TO901317 efficiently crossed the blood–brain barrier. Insoluble and soluble Aβ levels in the treated APP23 mice were reduced by 80% and 40%, respectively, compared with untreated animals. Amyloid precursor protein (APP) processing, however, was hardly changed by the compound, suggesting that the observed effects were instead mediated by Aβ disposal. Despite the profound effect on Aβ levels, spatial learning in the Morris water maze was only slightly improved by the treatment. ABCA1 (ATP-binding cassette transporter 1) and apolipoprotein E (ApoE) protein levels were increased and found to be primarily localized in astrocytes. Experiments using primary microglia demonstrated that medium derived from primary astrocytes exposed to TO901317 stimulated phagocytosis of fibrillar Aβ. Conditioned medium from TO901317-treated ApoE−/−or LXRα−/−astrocytes did not increase phagocytosis of Aβ. In APP23 mice, long-term treatment with TO901317 strongly increased the association of microglia and Aβ plaques. Short-term treatment of APP/PS1 mice with TO901317 also increased this association, which was dependent on the presence of LXRα and was accompanied by increased ApoE lipidation. Together, these data suggest that astrocytic LXRα activation and subsequent release of ApoE by astrocytes is critical for the ability of microglia to remove fibrillar Aβ in response to treatment with TO901317.

The orphan receptor LRH-1 and the oxysterol receptors LXRα and LXRβ are established transcriptional regulators of lipid metabolism that appear to control inflammatory processes. Here, we investigate the anti-inflammatory actions of these nuclear receptors in the hepatic acute phase response (APR). We report that selective synthetic agonists induce SUMOylation-dependent recruitment of either LRH-1 or LXR to hepatic APR promoters and prevent the clearance of the N-CoR corepressor complex upon cytokine stimulation. Investigations of the APR in vivo, using LXR knockout mice, indicate that the anti-inflammatory actions of LXR agonists are triggered selectively by the LXRβ subtype. We further find that hepatic APR responses in small ubiquitin-like modifier-1 (SUMO-1) knockout mice are increased, which is due in part to diminished LRH-1 action at APR promoters. Finally, we provide evidence that the metabolically important coregulator GPS2 functions as a hitherto unrecognized transrepression mediator of interactions between SUMOylated nuclear receptors and the N-CoR corepressor complex. Our study extends the knowledge of anti-inflammatory mechanisms and pathways directed by metabolic nuclear receptor–corepressor networks to the control of the hepatic APR, and implies alternative pharmacological strategies for the treatment of human metabolic diseases associated with inflammation.

Transcriptional coregulators, rather than ligand signals, are suspected to confer context and pathway specificity to nuclear receptor signaling, but the identity of such specifying coregulators and the underlying molecular mechanisms remain largely enigmatic. Here we address this issue in metabolic oxysterol receptor LXR pathways and describe the selective requirement of GPS2 for ABCG1 cholesterol transporter gene transcription and cholesterol efflux from macrophages. We implicate GPS2 in facilitating LXR recruitment to an ABCG1-specific promoter/enhancer unit upon ligand activation and identify functional links to histone H3K9 demethylation. We further describe fundamental differences between ABCG1 and ABCA1 with regard to GPS2 in relation to other coregulators, which are likely to apply to additional LXR-regulated genes. Our work identifies a coregulator-dependent epigenetic mechanism governing the access of a nuclear receptor to communicating regulatory regions in the genome. The pathway and coregulator selectivity of this mechanism implies pharmacological possibilities for the development of selective LXR agonists.

RAP250 is a coactivator for nuclear receptors as well as other transcription factors. Recent studies have established RAP250 as an essential coactivator for many important biological processes, but its exact mechanism of action is not fully understood. To identify novel proteins that can associate with RAP250, we used a yeast two-hybrid system to screen cDNA libraries and identified the intracellular mediators of transforming growth factor-beta (TGF-beta) response Smad2 and Smad3 as direct interacting proteins. We show that the interaction between RAP250 and Smad2/3 is dependent upon the second LXXLL interaction motif in RAP250 and the MH2 domain in Smad2 and Smad3. Mouse embryonic fibroblasts lacking RAP250 have reduced expression of the TGF-beta target gene PAI-1 after stimulation by TGF-beta when compared with wild type cells. Furthermore, we demonstrate a cross-talk between TGF-beta and liver X receptors (LXR) signaling pathways and show that stimulation of cells with TGF-beta and LXR agonists have a synergistic effect on the expression of the LXR target gene ABCG1. Our data identify RAP250 as a new coactivator in the TGF-beta signaling pathway that binds Smad2 and Smad3. Our data also suggest that the interaction between RAP250, Smad2, and Smad3 constitutes an important bridging mechanism linking LXR and TGF-beta signaling pathways.

The nuclear hormone receptors liver X receptor α (LXRα) (NR1H3) and LXRβ (NR1H2) are established regulators of cholesterol, lipid, and glucose metabolism and are attractive drug targets for the treatment of diabetes and cardiovascular disease. Adrenal steroid hormones including glucocorticoids and mineralocorticoids are known to interfere with glucose metabolism, insulin signaling, and blood pressure regulation. Here we present genome-wide expression profiles of LXR-responsive genes in both the adrenal and the pituitary gland. LXR activation in cultured adrenal cells inhibited expression of multiple steroidogenic genes and consequently decreased adrenal steroid hormone production. In addition, LXR agonist treatment elevated ACTH mRNA expression and hormone secretion from pituitary cells both in vitro and in vivo. Reduced expression of the glucocortioid-activating enzyme 11β-hydroxysteroid dehydrogenase 1 in pituitary cells upon LXR activation suggests blunting of the negative feedback of glucocorticoids by LXRs. In conclusion, LXRs independently interfere with the hypothalamic-pituitary-adrenal axis regulation at the level of the pituitary and the adrenal gland.

Thyroid hormones (THs) are essential for the regulation of development and metabolism in key organs. THs produce biological effects both by directly affecting gene expression through the interaction with nuclear receptors (genomic effects) and by activating protein kinases and/or ion channels (short-term effects). Such activations can be either direct, in the case of ion channels, or mediated by membrane or cytoplasmic receptors. Short-term-activated signalling pathways often play a role in the regulation of genomic effects. Several TH intermediate metabolites, which were previously considered without biological activity, have now been associated with a broad range of actions, mostly attributable to short-term effects. Here, we give an overview of the physiological roles and mechanisms of action of THs, focusing on the emerging position that TH metabolites are acquiring as important regulators of physiology and metabolism.

Nuclear hormone receptor liver X receptor-alpha (LXRα) plays a vital role in cholesterol homeostasis and is reported to play a role in adipose function and obesity although this is controversial. Conversely, mesenchymal stem cells (MSCs) are suggested to be a major source of adipocyte generation. Accordingly, we examined the role of LXRα in adipogenesis of MSCs. Adult murine MSCs (mMSCs) were isolated from wild type (WT) and LXR-null mice. Using WT mMSCs, we further generated cell lines stably overexpressing GFP-LXRα (mMSC/LXRα/GFP) or GFP alone (mMSC/GFP) by retroviral infection. Confluent mMSCs were differentiated into adipocytes by the established protocol. Compared with MSCs isolated from WT mice, MSCs from LXR-null mice showed significantly increased adipogenesis, as determined by lipid droplet accumulation and adipogenesis-related gene expression. Moreover, mMSCs stably overexpressing GFP-LXRα (mMSC/LXRα/GFP) exhibited significantly decreased adipogenesis compared with mMSCs overexpressing GFP alone (mMSC/GFP). Since Wnt/beta-catenin signaling is reported to inhibit adipogenesis, we further examined it. The LXR-null group showed significantly decreased Wnt expression accompanied by a decrease of cellular beta-catenin (vs. WT). The mMSC/LXRα/GFP group exhibited significantly increased Wnt expression accompanied by an increase of cellular beta-catenin (vs. mMSC/GFP). These data demonstrate that LXRα has an inhibitory effect on adipogenic differentiation in murine mesenchymal stem cells with Wnt/beta-catenin signaling. These results provide important insights into the pathophysiology of obesity and obesity related consequences such as metabolic syndrome and may identify potential therapeutic targets.

The oxysterol receptors, LXRs, have recently been shown to reduce cell and tumour growth in various model systems. Activation of LXRs could therefore provide a novel approach for treatment of cancers. Here we show that LXRβ is the main executor of the antiproliferative effect in human breast cancer cells. LXR inhibits the activation of growth factor-induced triggering of the PI3K-Akt pathway. Phosphorylation of several protein kinases in this pathway, including Akt and the PI3K itself, is reduced upon activation of LXR. Both mRNA and protein expression levels of the PTEN and PHLPPL protein phosphatases were induced by LXR and the amount of the second messenger PIP3 reduced—a pivotal activator signalling molecule in the PI3K. This suggest that the intracellular signalling cascade mediating proliferative cues from growth factors is the responsible mechanisms underlying the antiproliferative effects of LXR in human breast cancer cells. This provides novel and in-depth insights of how LXR works in cancer cells where the LXRs control the activity of intracellular signalling cascades that regulate proliferation.

Cholesterol precursors act as activators of the nuclear hormone receptor, liver X receptor (LXR). One of these LXR-activating ligands is meiosis activating sterol (MAS), which also induces resumption of meiosis in oocytes from mice in vitro. Whether LXR participates in the regulation of oocyte maturation and whether the expression of either one of the two paralogues of LXR (alpha and beta) affect fertility of mice has, however, not yet been clarified. Female mice lacking Lxra, Lxrb or both genes (Lxra(-/-), Lxrb(-/-) and Lxrab(-/-), respectively) conceive less frequently and have significantly fewer pups per litter as compared to wild type mice. Both Lxra and Lxrb mRNA were found to be expressed in mouse oocytes. The relative expression of, in particular, Lxrb was almost two orders of magnitude higher than in liver, brain and testis. A water-soluble LXR agonist caused naked oocytes, but not cumulus enclosed oocytes (CEO), from wild type mice to resume meiosis significantly more often than control oocytes. Follicle stimulating hormone (FSH) is a potent stimulator of meiosis in CEO from wild type mice, but was without effect in mice lacking both Lxr genes. Zymosterol, a MAS active substance, induced resumption of meiosis in oocytes from Lxrab(-/-) mice, but significantly less effectively than in oocytes from wild type mice. Taken together, LXRs seem to affect ovarian function, suggesting specific roles of cholesterol precursors in regulation of female reproduction.

Obesity and dyslipidemia are both risk factors for developing Type 2 diabetes and cardiovascular disease (CVD). These diseases have become a major health hazard in society; 177 million people suffered from diabetes worldwide in 2000 and this number will increase to at least 300 million by 2025. Type 2 diabetes and CVD are most probably of polygenetic origin and research has focused primarily on understanding regulation of lipid, cholesterol and glucose metabolism to elucidate the pathophysiology of these diseases. Liver X receptors are important regulators of fatty acid biosynthesis and cholesterol and glucose metabolism. They regulate transcription of key genes involved in several metabolic processes, including cytochrome P450 7A1, ATP binding cassette-lipid/cholesterol transporters, sterol regulatory element binding protein-1c and glucose transporter 4. Hence, liver X receptors promote reverse cholesterol transport, conversion of cholesterol to bile acids, reduced absorption of cholesterol in the intest...

The promotion of lipid efflux from macrophages is an important ATP binding cassette A1 (ABCA1)-mediated antiatherosclerotic mechanism that prevents peripheral tissues from foam cell accumulation. Statins exert beneficial antiatherosclerotic effects on cardiovascular disease correlated to the cholesterol-lowering properties and the pleiotropic activities. In this work, we investigated the ability of statins to modulate ABCA1-mediated lipid efflux from macrophages, where the protein expression was differently induced. Pitavastatin (0.1-10 microM) and compactin (10 microM) reduced both cholesterol and phospholipid efflux up to 60% from macrophages expressing ABCA1 upon treatment with 8-(4-chlorophenylthio)-cyclic AMP (cpt-cAMP), and this was secondary to a reduction of ABCA1 mRNA and protein content. Conversely, statins did not affect ABCA1 activity when the protein was up-regulated by 22-hydroxycholesterol/9-cis-retinoic acid or through cholesterol loading. Statin inhibition of lipid efflux induced by cpt-cAMP was reversed in the presence of mevalonate, 22-hydroxycholesterol, and cholesterol but not geranyl geraniol. In macrophages obtained from liver X receptor (LXR)-deficient mice, cpt-cAMP still promoted cholesterol efflux, but pitavastatin did not exert any effect. The present work shows that statins may inhibit ABCA1-mediated lipid efflux in macrophages only when ABCA1 protein expression is induced by cpt-cAMP and provides evidence that cAMP may activate ABCA1 independently of an increase of intracellular sterol synthesis but through at least two pathways: one independent of LXR and one involving an intracellular sterol(s) acting as LXR ligand(s). In addition, the lack of inhibitory effect on lipid efflux in cholesterol-loaded macrophages is likely to exclude a potential negative pleiotropic effect by statins.

Injection of myelin basic protein (MBP)-pulsed dendritic cells (DC) into healthy rats, as we reported before and observed in this study, did not induce clinical experimental allergic encephalomyelitis (EAE), but effectively protected the rats from subsequent EAE induction. The mechanisms by which MBP-pulsed DC mediate immune protection are not completely understood. In the present study, we mainly explored the dynamic change of cytokine and growth factor mRNA expression in spinal cords after subcutaneous injection of MBP-pulsed and unpulsed DC. The expression of interleukin (IL)-1, interferon-g and tumour necrosis factor-a as well as programmed death ligand (PDL)-1, PDL-2, signal transducer and activator of transcription (STAT)4, STAT6, matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinases (TIMP)-2 was increased on day 0 postimmunization (p.i.). The increase of IL-12 expression was observed on day 7 p.i., while the increase of IL-10 expression mainly occurred on day 14 p.i. Except downregulation of insulin-like growth factor-1, the expression of brain-derived neurotrophic factor, ciliary neurotrophic factor, fibroblast growth factor (FGF)-2 and platelet-derived growth factor (PDGF)-B/C as well as nerve growth factor receptor (NGF-R), FGF receptor, PDGF-R-a and b was elevated on day 0 p.i., while the increase of TIMP and NGF was observed on days 0 and 7 p.i. There were no significant differences on MMP-2, spinal cord-derived growth factor and PDGF-A mRNA expression. In line with the suppression of EAE induced by MBP-pulsed DC, the dynamic change of cytokines and growth factors in spinal cords should constitute a beneficial microenvironment against EAE.

Subjects from Sardinia, Italy, are relatively homogeneous compared to Swedes. Although ethnically distant, both populations have similarly high multiple sclerosis (MS) incidence rates. Pro- and anti-inflammatory cytokines and their receptors, signalling molecules and other immune response-associated factors might influence MS pathogenesis, though definite proof is missing. The study of populations with similar MS incidence but different genetic and environmental background could make possible the definition of factors that relate to such background differences. We selected untreated female MS patients from Sassari, Sardinia, and Stockholm, Sweden, and corresponding sexand age-matched healthy controls (HC), to study blood mononuclear cells (MNC) for mRNA expression of 20 immune response-related genes considered relevant in MS, employing real-time PCR. Higher expression of IL-12p40 mRNA was confined to MS from both Sassari and Stockholm, compared to corresponding HC. MS patients from Sassari, but not Stockholm, expressed higher TNF-a compared to corresponding HC. MS patients from Stockholm, but not Sassari, expressed higher IL-6. Indoleamine 2,3 dioxygenase (IDO), a molecule necessary in tolerance induction, was lower in MS from Stockholm compared to corresponding HC. This was not observed in Sassari. No differences were detected for other members of the IL-12 family, other Th1 and Th2 cytokines, and the signalling molecules Stat 4 and 6. The results corroborate a pro-inflammatory state in MS as reflected by high expression of IL-12, TNF-a and IL-6, although the extent of expression of TNF-a, IL-6 and IDO differs between strictly matched MS patients from different high-incidence areas. This might result from genetic and/or environmental differences. They may account for some of the discrepancies regarding immune response-related molecules previously reported in MS. In conclusion, a pro-inflammatory state exists in MS patients from Sassari as well as Stockholm. The changes of pro-inflammatory and other immune response-related variables differ however between the two MS populations. This may be attributed to the genetic and/or environmental background.

The nuclear receptors liver X receptor (LXR)alpha and LXRbeta are sensors of cholesterol metabolism and lipid biosynthesis. They have recently been found to be regulators of inflammatory cytokines, suppressors of hepatic glucose production, and involved in different cell-signaling pathways. LXRalpha is a target gene of the peroxisome proliferator-activated receptor-gamma, a target of drugs used in treating elevated levels of glucose seen in diabetes. Furthermore, insulin induces LXRalpha in hepatocytes, resulting in increased expression of lipogenic enzymes and suppression of key enzymes in gluconeogenesis, including PEPCK. LXR seems to have an important role in the regulation of glucocorticoid action and a role in the overall energy homeostasis suggested by its putative regulatory effect on leptin and uncoupling protein 1. The physiological roles of LXR indicate that it is an interesting potential target for drug treatment of diabetes.

Transforming acidic coiled-coil (TACC) proteins are hypothesized to play a role in normal cellular growth and differentiation and to be involved in centrosomal microtubule stabilization. Our current studies aim to delineate the expression pattern of TACC3 protein during cellular differentiation and in a variety of normal human tissues. TACC3 is known to be upregulated in differentiating erythroid progenitor cells following treatment with erythropoietin and is required for replication of hematopoietic stem cells. However, we demonstrate that a dramatic upregulation of TACC3 also occurs during the early differentiation of NIH 3T3-L1 cells into adipocytes and PC12 cells into neurons, indicating that TACC3 mediates cellular differentiation in several cell types. Using real-time PCR, we quantitated the mRNA levels of TACC3 compared to TACC1 and TACC2 in various human adult tissues. We observed the highest expression of TACC3 mRNA in testis, spleen, thymus and peripheral blood leukocytes, all tissues undergoing high rates of differentiation, and a lower level of expression in ovary, prostate, pancreas, colon, small intestine, liver and kidney. In contrast, TACC1 and TACC2 mRNA levels are more widespread. By immunohistochemistry, we confirm that the TACC3 protein localizes to differentiating cell types, including spermatocytes, oocytes, epithelial cells, bone marrow cells and lymphocytes. Thus, these observations are concordant with a basic role for TACC3 during early stages of differentiation in normal tissues.

Prenatal and postnatal RhD prophylaxis reduces the risk of RhD immunization in pregnancies of RhD-negative women. Based on the result from prenatal screening for the fetal RHD gene, prenatal RhD prophylaxis in Denmark is targeted to RhD-negative women who carry an RhD-positive fetus. Here, we present a 2-year evaluation of a nationwide prenatal RHD screening.Blood samples were drawn from RhD-negative women in gestational week 25. DNA was extracted from maternal plasma and analyzed for the RHD gene. The prenatal RHD results were compared with the serological typing of newborns in 12,668 pregnancies. Early compliance was assessed for 690 pregnancies.The sensitivity for the detection of fetal RHD was 99.9% (95% CI: 99.7-99.9%). Unnecessary recommendation of prenatal RhD prophylaxis was avoided in 97.3% of the women carrying an RhD-negative fetus. Fetuses that were seropositive for RhD were not detected in 11 pregnancies (0.087%). The sample uptake percentage was 84.2%, and the compliance for prenatal anti-D administration was 93.2%.The high sensitivity, maintained over 2 years, underlines the reliability of routine prenatal fetal RHD screening in RhD-negative pregnant women, specifically at 25 weeks of gestation. The remaining challenges are logistical and are related to program compliance.

Expression of the LXRalpha nuclear receptor in liver is predicted to affect cholesterol and lipid metabolism. Here we show that a short fragment from the LXRalpha gene promoter spanning the region from -144 to +43 relative to the mRNA initiation site can drive transcription of a reporter gene. Under basal conditions, in vitro DNase I footprinting demonstrated interaction between nuclear proteins and an NF1 recognition site in close vicinity to the transcriptional initiation. Both supershift, mutational analyses in EMSA and transfections provided evidence that the NF1 (nuclear factor I) transcription factor interacts with the LXRalpha promoter. All four members of the NF1 family were found to suppress the transcriptional activity indicating a general inhibitory effect on LXRalpha expression. A similar regulation by NF1 was also observed when using a fragment from the LXRalpha promoter extending up to position -3033 therefore giving the inhibitory effect of NF1 a significant impact on LXRalpha gene expression.

The liver X receptors (LXR)α and LXRβ are transcription factors belonging to the nuclear receptor family, which play a central role in metabolic homeostasis, being master regulators of key target genes in the glucose and lipid pathways. Wild-type (WT), LXRα−/−, and LXRβ−/−mice were fed a chow diet with (treated) or without (control) the synthetic dual LXR agonist GW3965 for 5 wk. GW3965 raised intrahepatic triglyceride (TG) level but, surprisingly, reduced serum TG level through the activation of serum lipase activity. The serum TG reduction was associated with a repression of both catecholamine-stimulated lipolysis and relative glucose incorporation into lipid in isolated adipocytes through activation of LXRβ. We also demonstrated that LXRα is required for basal (nonstimulated) adipocyte metabolism, whereas LXRβ acts as a repressor of lipolysis. On the contrary, in skeletal muscle (SM), the lipogenic and cholesterol transporter LXR target genes were markedly induced in WT and LXRα−/−mice and to a lesser extent in LXRβ−/−mice following treatment with GW3965. Moreover, TG content was reduced in SM of LXRβ−/−mice, associated with increased expression of the main TG-lipase genes Hsl and Atgl. Energy expenditure was increased, and a switch from glucose to lipid oxidation was observed. In conclusion, we provide evidence that LXR might be an essential regulator of the lipid balance between tissues to ensure appropriate control of the flux of fuel. Importantly, we show that, after chronic treatment with GW3965, SM becomes the target tissue for LXR activation, as opposed to liver, in acute treatment.

OR1 is a member of the superfamily of steroid/thyroid hormone nuclear receptors and recognizes DNA as a heterodimer with the 9-cis-retinoic acid receptor RXR (retinoid X receptor). The heterodimeric complex has been shown to be transcriptionally activatable by the RXR ligand as well as certain oxysterols via OR1, but to date uniquely also by heterodimerization itself. Recent studies on other members of the superfamily of nuclear receptors have led to the identification of a number of nuclear receptor-interacting proteins that mediate their regulatory effects on transcription. Here, we address the question of involvement of some of these cofactors in the three modes of activation by the OR1/RXRalpha complex. We show that in vitro the steroid receptor coactivator SRC-1 can be recruited by RXRalpha upon addition of its ligand, and to OR1 upon addition of 22(R)-OH-cholesterol, demonstrating that the latter can act as a direct ligand to OR1. Additionally, heterodimerization is sufficient to recruit SRC-1 to OR1/RXRalpha, indicating SRC-1 as a molecular mediator of dimerization-induced activation. In transfection experiments, coexpression of a nuclear receptor-interacting fragment of SRC-1 abolishes constitutive activation by OR1/RXRalpha, which can be restored by over-expression of full-length SRC-1. This constitutes evidence for an in vivo role of SRC-1 in dimerization-induced activation by OR1/RXRalpha. Additionally, we show that the nuclear receptor-interacting protein RIP140 binds in vitro to OR1 and RXRalpha with requirements distinct from those of SRC-1, and that binding of the two cofactors is competitive. Taken together, our results suggest a complex modulation of differentially induced transactivation by OR1/RXR coregulatory molecules.

MUC1 is a highly polymorphic mucin type glycoprotein expressed on the surface of many epithelia, including gastric mucosa, and is present in several body fluids and mucous secretions. A genetic polymorphism due to variation in length of a 60 bp tandemly repeated sequence domain constitutes more than half of the coding region of the glycoprotein. We demonstrated previously in a Portuguese population sample that the frequency of small MUC1 alleles is increased in patients with gastric carcinoma, suggesting that the possession of small MUC1 alleles confers increased risk for gastric carcinoma development. This finding raised the possibility that the very high prevalence of gastric carcinoma in Portugal could be partly due to a high frequency of small MUC1 alleles in the Portuguese population. In the present study we compared the MUC1 allele distribution in a population of Danish blood donors with the distribution in a population of Portuguese blood donors. The frequency of small MUC1 alleles was significantly higher in the Danish than in the Portuguese sample, thus failing to lend support to the hypothesis that a relatively higher frequency of the small MUC1 alleles might account for the high prevalence of gastric carcinoma in Portugal when compared to Denmark.

Reversing the escalating rate of obesity requires increased knowledge of the molecular mechanisms controlling energy balance. Liver X receptors (LXRs) and thyroid hormone receptors (TRs) are key physiological regulators of energetic metabolism. Analysing interactions between these receptors in the periphery has led to a better understanding of the mechanisms involved in metabolic diseases. However, no data is available on such interactions in the brain. We tested the hypothesis that hypothalamic LXR/TR interactions could co-regulate signalling pathways involved in the central regulation of metabolism. Using in vivo gene transfer we show that LXR activation by its synthetic agonist GW3965 represses the transcriptional activity of two key metabolic genes, Thyrotropin-releasing hormone (Trh) and Melanocortin receptor type 4 (Mc4r) in the hypothalamus of euthyroid mice. Interestingly, this repression did not occur in hypothyroid mice but was restored in the case of Trh by thyroid hormone (TH) treatment, highlighting the role of the triiodothyronine (T3) and TRs in this dialogue. Using shLXR to knock-down LXRs in vivo in euthyroid newborn mice, not only abrogated Trh repression but actually increased Trh transcription, revealing a potential inhibitory effect of LXR on the Hypothalamic-Pituitary-Thyroid axis. In vivo chromatin immunoprecipitation (ChIP) revealed LXR to be present on the Trh promoter region in the presence of T3 and that Retinoid X Receptor (RXR), a heterodimerization partner for both TR and LXR, was never recruited simultaneously with LXR. Interactions between the TR and LXR pathways were confirmed by qPCR experiments. T3 treatment of newborn mice induced hypothalamic expression of certain key LXR target genes implicated in metabolism and inflammation. Taken together the results indicate that the crosstalk between LXR and TR signalling in the hypothalamus centres on metabolic and inflammatory pathways.

Introduction The two oxysterol receptors, 'liver X receptors (LXRs)' LXRα and LXRβ, are amongst the emerging newer drug targets within the nuclear receptor family and targeting LXRs represents novel strategies needed for prevention and treatment of diseases where current therapeutics is inadequate. Areas covered This review discusses the current understanding of LXR biology with an emphasis on the molecular aspects of LXR signalling establishing their potential as drug targets. Recent advances of their transcriptional mechanisms in inflammatory pathways and their physiological roles in inflammation and immunity are described. Expert opinion The new discoveries of LXR-regulated inflammatory pathways have ignited new promises for LXRs as drug targets. The broad physiological roles of LXRs involve a high risk of unwanted side effects. Recent insights into LXR biology of the brain indicate a highly important role in neuronal development and a clinical trial testing an LXR agonist reported adverse neurological side effects. This suggests that drug development must focus on limiting the range of LXR signalling - possibly achieved through subtype, tissue specific, promoter specific or pathway specific activation of LXRs where a successful candidate drug must be carefully studied for its effect in the central nervous system.

Liver X receptors (LXRs) exert key functions in lipid homeostasis and in control of inflammation. In this study we have explored the impact of LXR activation on the macrophage response to the endogenous inflammatory cytokine IFN-γ. Transcriptional profiling studies demonstrate that ∼38% of the IFN-γ–induced transcriptional response is repressed by LXR activation in macrophages. LXRs also mediated inhibitory effects on selected IFN-γ–induced genes in primary microglia and in a model of IFN-γ–induced neuroinflammation in vivo. LXR activation resulted in reduced STAT1 recruitment to the promoters tested in this study without affecting STAT1 phosphorylation. A closer look into the mechanism revealed that SUMOylation of LXRs, but not the presence of nuclear receptor corepressor 1, was required for repression of the NO synthase 2 promoter. We have also analyzed whether IFN-γ signaling exerts reciprocal effects on LXR targets. Treatment with IFN-γ inhibited, in a STAT1-dependent manner, the LXR-dependent upregulation of selective targets, including ATP-binding cassette A1 (ABCA1) and sterol response element binding protein 1c. Downregulation of ABCA1 expression correlated with decreased cholesterol efflux to apolipoprotein A1 in macrophages stimulated with IFN-γ. The inhibitory effects of IFN-γ on LXR signaling did not involve reduced binding of LXR/retinoid X receptor heterodimers to target gene promoters. However, overexpression of the coactivator CREB-binding protein/p300 reduced the inhibitory actions of IFN-γ on the Abca1 promoter, suggesting that competition for CREB-binding protein may contribute to STAT1-dependent downregulation of LXR targets. The results from this study suggest an important level of bidirectional negative cross-talk between IFN-γ/STAT1 and LXRs with implications both in the control of IFN-γ–mediated immune responses and in the regulation of lipid metabolism.

We examined the function of the oxysterol receptors (LXRs) in inflammatory bowel disease (IBD) through studying dextran sodium sulfate (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice and by elucidating molecular mechanisms underlying their anti-inflammatory action. We observed that Lxr-deficient mice are more susceptible to colitis. Clinical indicators of colitis including weight loss, diarrhea and blood in feces appeared earlier and were more severe in Lxr-deficient mice and particularly LXRβ protected against symptoms of colitis. Addition of an LXR agonist led to faster recovery and increased survival. In contrast, Lxr-deficient mice showed slower recovery and decreased survival. In Lxr-deficient mice, inflammatory cytokines and chemokines were increased together with increased infiltration of immune cells in the colon epithelium. Activation of LXRs strongly suppressed expression of inflammatory mediators including TNFα. While LXRα had anti-inflammatory effects in CD11b(+) immune cell populations, LXRβ in addition had anti-inflammatory effects in colon epithelial cells. Lack of LXRβ also induced CD4(+)/CD3(+) immune cell recruitment to the inflamed colon. Expression of both LXRA and LXRB was significantly suppressed in inflamed colon from subjects with IBD compared with non-inflamed colon. Taken together, our observations suggest that the LXRs could provide interesting targets to reduce the inflammatory responses in IBD.

Liver X receptor (LXR) activation stimulates triglyceride (TG) accumulation in the liver. Several lines of evidence indicate that estradiol-17β (E2) reduces TG levels in the liver; however, the molecular mechanism underlying the E2 effect remains unclear. Here, we show that administration of E2 attenuated sterol regulatory element-binding protein (SREBP)-1 expression and TG accumulation induced by LXR activation in mouse liver. In estrogen receptor alpha (ERα) knockout (KO) and liver-specific ERα KO mice, E2 did not affect SREBP-1 expression or TG levels. Molecular analysis revealed that ERα is recruited to the SREBP-1c promoter through direct binding to LXR and inhibits coactivator recruitment to LXR in an E2-dependent manner. Our findings demonstrate the existence of a novel liver-dependent mechanism controlling TG accumulation through the nonclassical ER/LXR pathway. To confirm that a nonclassical ER/LXR pathway regulates ERα-dependent inhibition of LXR activation, we screened ERα ligands that were able to repress LXR activation without enhancing ERα transcriptional activity, and, as a result, we identified the phytoestrogen, phloretin. In mice, phloretin showed no estrogenic activity; however, it did reduce SREBP-1 expression and TG levels in liver of mice fed a high-fat diet to an extent similar to that of E2.We propose that ER ligands reduce TG levels in the liver by inhibiting LXR activation through a nonclassical pathway. Our results also indicate that the effects of ER on TG accumulation can be distinguished from its estrogenic effects by a specific ER ligand.

Studies in patients with microvascular angina (MA) or the cardiologic syndrome X have shown a hyperinsulinemic response to an oral glucose challenge, suggesting insulin resistance and a role for increased serum insulin in coronary microvascular dysfunction. The aim of the present study was to examine whether patients with MA are insulin-resistant. Nine patients with MA and seven control subjects were studied. All were sedentary and glucose-tolerant. Coronary arteriography was normal in all participants, and exercise-induced coronary ischemia was demonstrated in all MA patients. A euglycemic, hyperinsulinemic clamp was performed in combination with indirect calorimetry. Biopsy of vastus lateralis muscle was taken in the basal state and after 4 hours of euglycemia and hyperinsulinemia (2 mU · kg−1 · min−1). The fasting level of “true” serum insulin was significantly higher (43 ± 6 v 22 ± 3 pmol/L, P < .02) and the rate of insulin-stimulated glucose disposal to peripheral tissues was lower in patients with MA (13.4 ± 1.0 v 18.2 ± 1.4 mg · kg fat-free mass [FFM]−1 · min−1, P < .02) due to a decrease in nonoxidative glucose metabolism (8.4 ± 0.9 v 12.5 ± 1.3 mg · kg FFM−1 · min−1, P < .02). No difference was found in glucose or lipid oxidation rates between the two groups. In patients with MA, as well as in the pooled group of all participants, a positive correlation was demonstrated between the insulin-stimulated increment in fractional activity of skeletal muscle glycogen synthase (GS) and the increment in nonoxidative glucose metabolism after in vivo insulin exposure (MA: r = .73, P = .03; all participants: r = .73, P = .005), indicating a reduced in vivo activation of GS in MA patients. In conclusion, (1) MA is part of the insulin resistance syndrome, and (2) the insulin resistance is predominantly localized to the glycogen synthesis pathway in skeletal muscle tissue.