Histone methyltransferase Smyd1 regulates mitochondrial energetics in the heart.

Smyd1, a muscle-specific histone methyltransferase, has established roles in skeletal and cardiac muscle development, but its role in the adult heart remains poorly understood. Our prior work demonstrated that cardiac-specific deletion of Smyd1 in adult mice (Smyd1-KO) leads to hypertrophy and heart failure. Here we show that down-regulation of mitochondrial energetics is an early event in these Smyd1-KO mice preceding the onset of structural abnormalities. This early impairment of mitochondrial energetics in Smyd1-KO mice is associated with a significant reduction in gene and protein expression of PGC-1α, PPARα, and RXRα, the master regulators of cardiac energetics. The effect of Smyd1 on PGC-1α was recapitulated in primary cultured rat ventricular myocytes, in which acute siRNA-mediated silencing of Smyd1 resulted in a greater than twofold decrease in PGC-1α expression without affecting that of PPARα or RXRα. In addition, enrichment of histone H3 lysine 4 trimethylation (a mark of gene activation) at the PGC-1α locus was markedly reduced in Smyd1-KO mice, and Smyd1-induced transcriptional activation of PGC-1α was confirmed by luciferase reporter assays. Functional confirmation of Smyd1's involvement showed an increase in mitochondrial respiration capacity induced by overexpression of Smyd1, which was abolished by siRNA-mediated PGC-1α knockdown. Conversely, overexpression of PGC-1α rescued transcript expression and mitochondrial respiration caused by silencing Smyd1 in cardiomyocytes. These findings provide functional evidence for a role of Smyd1, or any member of the Smyd family, in regulating cardiac energetics in the adult heart, which is mediated, at least in part, via modulating PGC-1α.

Silencing Retinoid X Receptor Alpha Expression Enhances Early-Stage Hepatitis B Virus Infection In Cell Cultures.

Multiple steps of the life cycle of hepatitis B virus (HBV) are known to be coupled to hepatic metabolism. However, the details of involvement of the hepatic metabolic milieu in HBV infection remain incompletely understood. Hepatic lipid metabolism is controlled by a complicated transcription factor network centered on retinoid X receptor alpha (RXRα). Here, we report that RXRα negatively regulates HBV infection at an early stage in cell cultures. The RXR-specific agonist bexarotene inhibits HBV in HepG2 cells expressing the sodium taurocholate cotransporting polypeptide (NTCP) (HepG2-NTCP), HepaRG cells, and primary Tupaia hepatocytes (PTHs); reducing RXRα expression significantly enhanced HBV infection in the cells. Transcriptome sequencing (RNA-seq) analysis of HepG2-NTCP cells with a disrupted RXRα gene revealed that reduced gene expression in arachidonic acid (AA)/eicosanoid biosynthesis pathways, including the AA synthases phospholipase A2 group IIA (PLA2G2A), is associated with increased HBV infection. Moreover, exogenous treatment of AA inhibits HBV infection in HepG2-NTCP cells. These data demonstrate that RXRα is an important cellular factor in modulating HBV infection and implicate the participation of AA/eicosanoid biosynthesis pathways in the regulation of HBV infection.IMPORTANCE Understanding how HBV infection is connected with hepatic lipid metabolism may provide new insights into virus infection and its pathogenesis. By a series of genetic studies in combination with transcriptome analysis and pharmacological assays, we here investigated the role of cellular retinoid X receptor alpha (RXRα), a crucial transcription factor for controlling hepatic lipid metabolism, in de novo HBV infection in cell cultures. We found that silencing of RXRα resulted in elevated HBV covalently closed circular DNA (cccDNA) formation and viral antigen production, while activation of RXRα reduced HBV infection efficiency. Our results also showed that silencing phospholipase A2 group IIA (PLA2G2A), a key enzyme of arachidonic acid (AA) synthases, enhanced HBV infection efficiency in HepG2-NTCP cells and that exogenous AA treatment reduced de novo HBV infection in the cells. These findings unveil RXRα as an important cellular factor in modulating HBV infection and may point to a new strategy for host-targeted therapies against HBV.

Reduction in promotor methylation utilizing EGCG (epigallocatechin-3-gallate) restores RXRα expression in human colon cancer cells.

Silencing of regulatory genes through hypermethylation of CpG islands is an important mechanism in tumorigenesis. In colon cancer, RXRα, an important dimerization partner with other nuclear transcription factors, is silenced through this mechanism. We previously found that colon tumors in ApcMin/+ mice had diminished levels of RXRα protein and expression levels of this gene were restored by treatment with a green tea intervention, due to reduced promoter methylation of RXRα. We hypothesized that CIMP+ cell lines, which epigenetically silence key regulatory genes would also evidence silencing of RXRα and EGCG treatment would restore its expression. We indeed found EGCG to restore RXRα activity levels in the human cell lines, in a dose dependent manner and reduced RXRα promoter methylation. EGCG induced methylation changes in several other colon cancer related genes but did not cause a decrease in global methylation. Numerous epidemiological reports have shown the benefits of green tea consumption in reducing colon cancer risk but to date no studies have shown that the risk reduction may be related to the epigenetic restoration by tea polyphenols. Our results show that EGCG modulates the reversal of gene silencing involved in colon carcinogenesis providing a possible avenue for colon cancer prevention and treatment.

RXRα is upregulated in first trimester endometrial glands of spontaneous abortions unlike LXR and PPARγ.

Nuclear receptors are necessary for uterine invasion of the trophoblast and therefore important for maintaining a viable pregnancy. The aim of this study was to investigate the expression pattern and frequency of LXR, PPARγ and RXRα under physiological circumstances and in spontaneous abortions in endometrial glands and decidual tissue cells. A total of 28 (14 physiologic pregnancies/14 spontaneous abortion) human pregnancies in first trimester were analysed for expression of the nuclear receptors LXR, RXRα and PPARγ. Expression changes were evaluated by immunohistochemistry in decidual tissue and endometrial glands of the decidua. RXRα expression was up-regulated in the endometrial glands of spontaneous abortion (P<0.015). Similar up regulation of RXRα was found in decidual tissue (P<0.05). LXR and PPARγ expression was unchanged in spontaneous abortion. Via Correlation analysis we found a trend to positive correlation of LXR and PPARγ (Spearman correlation coefficient r=0.56 P=0.07) in endometrial glands. In decidual tissue, we found significant negative correlation in the control group, for the combination of RXRα and PPARγ (Spearman correlation coefficient r=0.913, P=0.03). Our data show that RXRα expression is increased in miscarriage in endometrial glands and correlation analysis showed that negative correlation between RXRα and PPARγ disappears in miscarriage. This shift is supposable responsible for the loss of regular function in trophoblast and embryonic tissue.

Pregnane X Receptor Expression in Human Pancreatic Adenocarcinoma: Associations With Clinicopathologic Parameters, Tumor Proliferative Capacity, Patients' Survival, and Retinoid X Receptor Expression.

OBJECTIVES: Pregnane X receptor (PXR) has been involved in human malignancy, either by directly affecting carcinogenesis or by inducing drug-drug interactions and chemotherapy resistance. The present study aimed to assess the clinical significance of PXR in pancreatic adenocarcinoma. METHODS: Pregnane X receptor and its heterodimers' PXR/retinoid X receptor α (RXR-α), RXR-ß, and RXR-γ expression were assessed immunohistochemically on tumoral samples from 55 pancreatic adenocarcinoma patients and were associated with clinicopathologic parameters, tumor proliferative capacity, and patients' survival. RESULTS: Enhanced PXR expression was noted in 24 (43.6%) of 55 pancreatic adenocarcinoma cases. Pancreatic adenocarcinoma patients presenting increased histological grade of tumor differentiation showed a significant increased incidence of elevated PXR expression (P = 0.023). Enhanced PXR/RXR-ß expression was significantly associated with smaller tumor size and earlier clinical stage (P = 0.005 and P = 0.003, respectively). Elevated PXR/RXR-γ expression was significantly associated with smaller tumor size and earlier clinical stage (P = 0.012 and P = 0.014, respectively) and borderline with the absence of lymph node metastases (P = 0.056). In addition, pancreatic adenocarcinoma patients presenting enhanced PXR/RXR-γ expression showed marginally longer survival times compared with those with decreased expression (log-rank test, P = 0.053). CONCLUSIONS: This study supported evidence that PXR and its copartners' overexpression may be associated with favorable clinicopathologic parameters and better outcome in pancreatic adenocarcinoma.

Retinoid X receptor α overexpression alleviates mitochondrial dysfunction-induced insulin resistance through transcriptional regulation of insulin receptor substrate 1.

Mitochondrial dysfunction is associated with insulin resistance and diabetes. We previously showed that retinoid X receptor α (RXRα) played an important role in transcriptional regulation of oxidative phosphorylation (OXPHOS) genes in cells with mitochondrial dysfunction caused by mitochondrial DNA mutation. In this study, we investigated whether mitochondrial dysfunction induced by incubation with OXPHOS inhibitors affects insulin receptor substrate 1 (IRS1) mRNA and protein levels and whether RXRα activation or overexpression can restore IRS1 expression. Both IRS1 and RXRα protein levels were significantly reduced when C2C12 myotubes were treated with the OXPHOS complex inhibitors, rotenone and antimycin A. The addition of RXRα agonists, 9-cis retinoic acid (9cRA) and LG1506, increased IRS1 transcription and protein levels and restored mitochondrial function, which ultimately improved insulin signaling. RXRα overexpression also increased IRS1 transcription and mitochondrial function. Because RXRα overexpression, knock-down, or activation by LG1506 regulated IRS1 transcription mostly independently of mitochondrial function, it is likely that RXRα directly regulates IRS1 transcription. Consistent with the hypothesis, we showed that RXRα bound to the IRS1 promoter as a heterodimer with peroxisome proliferator-activated receptor δ (PPARδ). These results suggest that RXRα overexpression or activation alleviates insulin resistance by increasing IRS1 expression.

Flow cytometry detection of vitamin D receptor changes during vitamin D treatment in Crohn's disease.

Crohn's disease (CD) is a chronic inflammatory disease associated with a dysregulated T cell response towards intestinal microflora. Vitamin D has immune modulatory effects on T cells through the nuclear vitamin D receptor (VDR) in vitro. It is unclear how oral vitamin D treatment affects VDR expression. The aim of this study was to establish a flow cytometry protocol, including nuclear and cytoplasmic VDR expression, and to investigate the effects of vitamin D treatment on T cell VDR expression in CD patients. The flow cytometry protocol for VDR staining was developed using the human acute monocytic leukaemia cell line (THP-1). The protocol was evaluated in anti-CD3/CD28-stimulated peripheral blood mononuclear cells (PBMCs) from vitamin D3- (n = 9) and placebo-treated (n = 9) CD patients. Anti-VDR-stained PBMCs were examined by flow cytometry, and their cytokine production was determined by cytokine bead array. VDR, CYP27B1 and RXRα mRNA expression levels in CD4(+) T cells were measured by quantitative reverse transcriptase polymerase chain reaction. The flow cytometry protocol enabled detection of cytoplasmic and nuclear VDR expression. The results were confirmed by confocal microscopy and supported by correlation with VDR mRNA expression. VDR expression in CD4(+) T cells increased following stimulation. This VDR up-regulation was inhibited with 30% by vitamin D treatment compared to placebo in CD patients (P = 0027). VDR expression was correlated with in-vitro interferon-γ production in stimulated PBMCs (P = 0.01). Flow cytometry is a useful method with which to measure intracellular VDR expression. Vitamin D treatment in CD patients reduces T cell receptor-mediated VDR up-regulation.

Long-distance retinoid signaling in the zebra finch brain.

All-trans retinoic acid (ATRA), the main active metabolite of vitamin A, is a powerful signaling molecule that regulates large-scale morphogenetic processes during vertebrate embryonic development, but is also involved post-natally in regulating neural plasticity and cognition. In songbirds, it plays an important role in the maturation of learned song. The distribution of the ATRA-synthesizing enzyme, zRalDH, and of ATRA receptors (RARs) have been described, but information on the distribution of other components of the retinoid signaling pathway is still lacking. To address this gap, we have determined the expression patterns of two obligatory RAR co-receptors, the retinoid X receptors (RXR) α and γ, and of the three ATRA-degrading cytochromes CYP26A1, CYP26B1, and CYP26C1. We have also studied the distribution of zRalDH protein using immunohistochemistry, and generated a refined map of ATRA localization, using a modified reporter cell assay to examine entire brain sections. Our results show that (1) ATRA is more broadly distributed in the brain than previously predicted by the spatially restricted distribution of zRalDH transcripts. This could be due to long-range transport of zRalDH enzyme between different nuclei of the song system: Experimental lesions of putative zRalDH peptide source regions diminish ATRA-induced transcription in target regions. (2) Four telencephalic song nuclei express different and specific subsets of retinoid-related receptors and could be targets of retinoid regulation; in the case of the lateral magnocellular nucleus of the anterior nidopallium (lMAN), receptor expression is dynamically regulated in a circadian and age-dependent manner. (3) High-order auditory areas exhibit a complex distribution of transcripts representing ATRA synthesizing and degrading enzymes and could also be a target of retinoid signaling. Together, our survey across multiple connected song nuclei and auditory brain regions underscores the prominent role of retinoid signaling in modulating the circuitry that underlies the acquisition and production of learned vocalizations.

Retinoid X receptor α attenuates host antiviral response by suppressing type I interferon.

The retinoid X receptor α (RXRα), a key nuclear receptor in metabolic processes, is downregulated during host antiviral response. However, the roles of RXRα in host antiviral response are unknown. Here we show that RXRα overexpression or ligand activation increases host susceptibility to viral infections in vitro and in vivo, while Rxra-/- or antagonist treatment reduces infection by the same viruses. Consistent with these functional studies, ligand activation of RXR inhibits the expression of antiviral genes including type I interferon (IFN) and Rxra-/- macrophages produce more IFNß than WT macrophages in response to polyI:C stimulation. Further results indicate that ligand activation of RXR suppresses the nuclear translocation of ß-catenin, a co-activator of IFNß enhanceosome. Thus, our studies have uncovered a novel RXR-dependent innate immune regulatory pathway, suggesting that the downregulation of RXR expression or RXR antagonist treatment benefits host antiviral response, whereas RXR agonist treatment may increase the risk of viral infections.

Impact of parenteral nutrition versus fasting on hepatic bile acid production and transport in a rabbit model of prolonged critical illness.

BACKGROUND: Cholestatic liver dysfunction frequently occurs during critical illness. Administration of parenteral nutrition (PN) is thought to aggravate this. Underlying mechanisms are not clear. METHODS: In a burn model of prolonged critical illness, rabbits were randomized to a nutritional strategy either accepting caloric deficits (fasted, n = 11) or covering caloric needs by PN (fed, n = 10). At baseline and after 7 days of critical illness, markers of hepatotoxicity, circulating bile acids, and the hepatobiliary transport system were studied. RESULTS: Fasted animals had lower circulating alanine aminotransferase/aspartate aminotransferase levels than did the fed animals at day 7. Compared with baseline values, fed animals displayed lower serum unconjugated cholic acid (CA) and deoxycholic acid (DCA) levels. Unconjugated DCA remained unaltered in fasted animals. Unconjugated lithocholic acid was increased comparably in all animals, whereas hyodeoxycholic acid was not altered. In contrast, fasting induced a shift from unconjugated CA and DCA to glyco-CA and glyco-DCA. Total bile acids did not correlate with the bile acid-producing enzyme CYP7A1, but with the basolateral efflux transporter MRP3. Fasting increased protein expression of the basolateral (MRP3) and the canalicular (BSEP) transporter, whereas the canalicular efflux pump MRP2 was suppressed. Gene expression levels of the nuclear receptor farnesoid X receptor were lower with fasting and correlated inversely with MRP3. The heterodimer partner of farnesoid X receptor, retinoid X receptor α, was increased with fasting and correlated positively with MRP3. CONCLUSIONS: During prolonged critical illness, withholding PN improved markers for hepatocyte injury and accentuated bile acid transport toward the blood. This suggests that the latter is an adaptive rather than a dysfunctional feedback to illness.

Retinoid X receptor agonists impair arterial mononuclear cell recruitment through peroxisome proliferator-activated receptor-γ activation.

Mononuclear cell migration into the vascular subendothelium constitutes an early event of the atherogenic process. Because the effect of retinoid X receptor (RXR)α on arterial mononuclear leukocyte recruitment is poorly understood, this study investigated whether RXR agonists can affect this response and the underlying mechanisms involved. Decreased RXRα expression was detected after 4 h stimulation of human umbilical arterial endothelial cells with TNF-α. Interestingly, under physiological flow conditions, TNF-α-induced endothelial adhesion of human mononuclear cells was concentration-dependently inhibited by preincubation of the human umbilical arterial endothelial cells with RXR agonists such as bexarotene or 9-cis-retinoid acid. RXR agonists also prevented TNF-α-induced VCAM-1 and ICAM-1 expression, as well as endothelial growth-related oncogene-α and MCP-1 release. Suppression of RXRα expression with a small interfering RNA abrogated these responses. Furthermore, inhibition of MAPKs and NF-κB pathways were involved in these events. RXR agonist-induced antileukocyte adhesive effects seemed to be mediated via RXRα/peroxisome proliferator-activated receptor (PPAR)γ interaction, since endothelial PPARγ silencing abolished their inhibitory responses. Furthermore, RXR agonists increased RXR/PPARγ interaction, and combinations of suboptimal concentrations of both nuclear receptor ligands inhibited TNF-α-induced mononuclear leukocyte arrest by 60-65%. In vivo, bexarotene dose-dependently inhibited TNF-α-induced leukocyte adhesion to the murine cremasteric arterioles and decreased VCAM-1 and ICAM-1 expression. Therefore, these results reveal that RXR agonists can inhibit the initial inflammatory response that precedes the atherogenic process by targeting different steps of the mononuclear recruitment cascade. Thus, RXR agonists may constitute a new therapeutic tool in the control of the inflammatory process associated with cardiovascular disease.

Seasonal variation in expression of markers in the vitamin D pathway in prostate tissue.

PURPOSE: Recent studies suggest variation in genes along the vitamin D pathway, as well as vitamin D receptor (VDR) protein levels, may be associated with prostate cancer. As serum vitamin D levels vary by season, we sought to determine whether the expression of genes on the vitamin D pathway, assessed in prostate tumor tissue, do the same. METHODS: Our study incorporates mRNA expression data from 362 men in the Swedish Watchful Waiting cohort, diagnosed between 1977 and 1999, and 106 men enrolled in the US Physicians' Health Study (PHS) diagnosed between 1983 and 2004. We also assayed for VDR protein expression among 832 men in the PHS and Health Professionals Follow-up Study cohorts. Season was characterized by date of initial tissue specimen collection categorically and by average monthly ultraviolet radiation levels. One-way analysis of variance was used to examine variation in the expression levels of six genes on the vitamin D pathway-VDR, GC, CYP27A1, CYP27B1, RXRα, CYP24A1-and VDR protein by season, adjusted for age at diagnosis and Gleason grade. Variation was also examined separately among lethal and nonlethal cases. RESULTS: Tumor expression levels of the six genes did not vary significantly by season of tissue collection. No consistent patterns emerged from subgroup analyses by lethal versus nonlethal cases. CONCLUSIONS: Unlike circulating levels of 25(OH) vitamin D, expression levels of genes on the vitamin D pathway and VDR protein did not vary overall by season of tissue collection. Epidemiological analyses of vitamin D gene expression may not be biased by seasonality.

Tangeretin stimulates glucose uptake via regulation of AMPK signaling pathways in C2C12 myotubes and improves glucose tolerance in high-fat diet-induced obese mice.

Although the flavonoid tangeretin (5, 6, 7, 8, 4'-pentamethoxyflavone) is known to possess beneficial health effects, the anti-diabetic effects and the mechanism of action have not been elucidated. Treatment with 100 µM tangeretin significantly increased the uptake of 2-NBDG in C2C12 myotubes. We also found that AMPK and AS160 were markedly phosphorylated by tangeretin treatment. In addition, pretreatment with an AMPK inhibitor significantly abrogated tangeretin-stimulated AS160 phosphorylation, glucose uptake, and Glut4 translocation from the cytosol to the plasma membrane. Furthermore, disruption of AMPK using siRNA transfection prevented the glucose uptake stimulated by tangeretin. We also examined the anti-diabetic properties of tangeretin in mice on HFD. Administration of HFD plus 200 mg/kg of tangeretin significantly altered weight gain, glucose tolerance, total cholesterol levels, and the secretion of adipocytokines, such as adiponectin, leptin, resistin, IL-6, and MCP-1. Moreover, AMPK was activated by 200 mg/kg of tangeretin in mouse muscle tissue, as expected from the cell system. These results suggest that tangeretin exerts anti-diabetic effects in both cell culture and mouse models, and these effects are necessary for activating AMPK.

Retinoid X receptor alpha (RXRα) and peroxisome proliferator-activated receptor gamma (PPARγ) expression in breast cancer: an immunohistochemical study.

BACKGROUND/AIM: The role of retinoid X receptor alpha (RXRα) and peroxisome proliferator-activated receptor gamma (PPARγ) in breast cancer has been well studied in vitro. The aim of the study was to assess the presence of these molecules in human breast cancer specimens and correlate them with major clinicopathological features. PATIENTS AND METHODS: Tissue sections from 82 breast cancer cases clustered according to histological grade, lymph node (LN) and hormone receptor (HR) status were assessed by immunohistochemistry for RXRα and PPARγ. RESULTS: RXRα was found to be strongly and moderately expressed in 11 (14.10%) and 33 (42.31%) cases, respectively. PPARγ was found to be strongly and moderately expressed in 33 (41.25%) and 25 (31.25%) cases, respectively. Only RXRα expression was inversely correlated with histological grade. Surprisingly, significantly elevated PPARγ expression was found in cases with positive LN status. Survival analysis did not yield significant results. CONCLUSION: Our data support the current thesis of RXRα being a potential target for feature molecular interventions.

Alteration of hepatic nuclear receptor-mediated signaling pathways in hepatitis C virus patients with and without a history of alcohol drinking.

UNLABELLED: The current study tests a hypothesis that nuclear receptor signaling is altered in chronic hepatitis C patients and that the altered pattern is specific to alcohol drinking history. The expression of a panel of more than 100 genes encoding nuclear receptors, coregulators, and their direct/indirect targets was studied in human livers. Gene expression pattern was compared between 15 normal donor livers and 23 hepatitis C virus (HCV) genotype 1-positive livers from patients without a drinking history (matched for age, sex, and body mass index). HCV infection increased the expression of nuclear receptors small heterodimer partner and constitutive androstane receptor (CAR) as well as genes involved in fatty acid trafficking, bile acid synthesis and uptake, and inflammatory response. However, the expression of retinoid X receptor (RXR) α, peroxisomal proliferator-activated receptor (PPAR) α and ß as well as steroid regulatory element-binding protein (SREBP)-1c was decreased in HCV-infected livers. Gene expression pattern was compared in chronic hepatitis C patients with and without a drinking history. Alcohol drinking increased the expression of genes involved in fatty acid uptake, trafficking, and oxidation, but decreased the expression of genes responsible for gluconeogenesis. These changes were consistent with reduced fasting plasma glucose levels and altered expression of upstream regulators that include RXRα, PPARα, and CAR. The messenger RNA levels of fibroblast growth factor 21, interleukin-10, and fatty acid synthase, which are all regulated by nuclear receptors, showed independent correlation with hepatic HCV RNA levels. CONCLUSION: Our findings suggest that those genes and pathways that showed altered expression could potentially be therapeutic targets for HCV infection and/or alcohol drinking-induced liver injury.

Suppression of retinoid X receptor alpha and aberrant ß-catenin expression significantly associates with progression of colorectal carcinoma.

To investigate retinoid X receptor alpha (RXRα) and ß-catenin expression and their relationship with the clinicopathological features of colorectal carcinoma (CRC). Real-time PCR and western blot analyses revealed that ß-catenin and RXRα expression at both mRNA and protein levels in four pairs of fresh CRC and adjacent non-tumour tissues (ANT) dramatically was increased and decreased in CRC compared with ANT, respectively. Furthermore, RXRα expression at both mRNA and protein levels was downregulated in higher histological grade CRC. Immunohistochemistry staining in 120 cases of CRC and 60 cases of lymph node metastatic carcinoma of CRC showed that RXRα expression was significantly suppressed in CRC compared with ANT (P<0.001) and low expression of RXRα in CRC was significantly associated with histological grade (P<0.001), TNM stage (P=0.022) and N classification (P=0.002). The aberrant (accumulated cytoplasm or/and nuclei) expression of ß-catenin was higher in CRC than that in ANT (P<0.001) and associated with histological grade (P=0.001) and N classification (P=0.002). Moreover, there was a close relationship between low RXRα expression and aberrant ß-catenin expression in CRC (P=0.032). Taken together with our previous study, aberrant ß-catenin expression upregulated by suppression of RXRα may play a crucial role in pathogenesis and progression of CRC.

Activation of the aryl hydrocarbon receptor induces hepatic steatosis via the upregulation of fatty acid transport.

The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix/Per-ARNT-Sim domain transcription factor, which is activated by various xenobiotic ligands. AHR is known to be abundant in liver tissue and to be associated with hepatic steatosis. However, it has not yet been elucidated how the activation of AHR promotes hepatic steatosis. The aim of this study is to clarify the role of AHR in hepatic steatosis. The intraperitoneal injection of 3-methylcholanthrene (3MC), a potent AHR ligand, into C57BL/6J mice significantly increased the levels of triglycerides and six long-chain monounsaturated fatty acids in the livers of mice, resulting in hepatic microvesicular steatosis. 3MC significantly enhanced the expression level of fatty acid translocase (FAT), a factor regulating the uptake of long-chain fatty acids into hepatocytes, in the liver. In an in vitro experiment using human hepatoma HepG2 cells, 3MC increased the expression level of FAT, and the downregulation of AHR by AHR siRNA led to the suppression of 3MC-induced FAT expression. In addition, the mRNA level of peroxisome proliferator-activated receptor (PPAR) α, an upstream factor of FAT, was increased in the livers of 3MC-treated mice. Taking together, AHR activation induces hepatic microvesicular steatosis by increasing the expression level of FAT.

Silibinin can induce differentiation as well as enhance vitamin D3-induced differentiation of human AML cells ex vivo and regulates the levels of differentiation-related transcription factors.

Induction of terminal differentiation is a conceptually attractive approach for the therapy of neoplastic diseases. Although vitamin D derivatives (deltanoids) can induce differentiation of AML cells in vitro, so far deltanoids have not been successfully brought to the clinic, due to the likelihood of life-threatening hypercalcemia. Here, we incubated freshly obtained blood cells from patients with AML with a plant antioxidant (PAOx), silibinin (SIL), alone or together with a deltanoid. Twenty patients with AML (all subtypes except M3) were available for this study, and in 14 (70%), SIL (60 µM) either induced differentiation ex vivo, or enhanced differentiation induced by deltanoids, or both. Interestingly, SIL acting alone induced differentiation only in cases in which chromosome aberrations could not be detected. In eleven samples sufficient material was available for a limited analysis of the underlying events. Quantitative RT-PCR showed that differentiation markers were upregulated at the mRNA level by both SIL and deltanoids, suggesting that intracellular signaling pathways upstream of transcription factors (TFs) were activated by these agents. Western analysis for proteins which function as TFs in deltanoid-induced monocytic differentiation, such as members of Jun and C/EBP families, surprisingly demonstrated that SIL upregulated all these TFs in the cases tested. This suggests that although the presence of SIL may not always be sufficient to induce differentiation, it can serve as a differentiation enabling factor for blasts obtained from a large proportion of patients with AML. Thus, SIL/deltanoid combinations warrant further consideration as preventive/therapeutic regimens in human leukaemia.

Peroxisome proliferator-activated receptor gamma ligands enhance human B cell antibody production and differentiation.

Protective humoral immune responses critically depend on the optimal differentiation of B cells into Ab-secreting cells. Because of the important role of Abs in fighting infections and in successful vaccination, it is imperative to identify mediators that control B cell differentiation. Activation of B cells through TLR9 by CpG-DNA induces plasma cell differentiation and Ab production. Herein, we examined the role of the peroxisome proliferator-activated receptor (PPAR)gamma/RXRalpha pathway on human B cell differentiation. We demonstrated that activated B cells up-regulate their expression of PPARgamma. We also show that nanomolar levels of natural (15-deoxy-Delta(12,14)-prostaglandin J(2)) or synthetic (rosiglitazone) PPARgamma ligands enhanced B cell proliferation and significantly stimulated plasma cell differentiation and Ab production. Moreover, the addition of GW9662, a specific PPARgamma antagonist, abolished these effects. Retinoid X receptor (RXR) is the binding partner for PPARgamma and is required to produce an active transcriptional complex. The simultaneous addition of nanomolar concentrations of the RXRalpha ligand (9-cis-retinoic acid) and PPARgamma ligands to CpG-activated B cells resulted in additive effects on B cell proliferation, plasma cell differentiation, and Ab production. Furthermore, PPARgamma ligands alone or combined with 9-cis-retinoic acid enhanced CpG-induced expression of Cox-2 and the plasma cell transcription factor BLIMP-1. Induction of these important regulators of B cell differentiation provides a possible mechanism for the B cell-enhancing effects of PPARgamma ligands. These new findings indicate that low doses of PPARgamma/RXRalpha ligands could be used as a new type of adjuvant to stimulate Ab production.

The basic helix-loop-helix proteins differentiated embryo chondrocyte (DEC) 1 and DEC2 function as corepressors of retinoid X receptors.

The basic helix-loop-helix proteins differentiated embryo chondrocyte 1 (DEC1) and DEC2 are involved in circadian rhythm control. Because the metabolism of dietary nutrients has been linked to circadian regulation, we examined the effect of DEC1 and DEC2 on the function of the metabolite-sensing nuclear receptors, ligand-dependent transcription factors, including retinoid X receptor (RXR) and liver X receptor (LXR). Transfection assays showed that DEC1 and DEC2 repressed ligand-dependent transactivation by RXR. Knockdown of endogenous DEC1 and DEC2 expression with small interfering RNAs augmented ligand-dependent RXRalpha transactivation. DEC1 and DEC2 interacted directly with RXRalpha, and ligand addition enhanced their association. DEC1 and DEC2 modified interaction of RXRalpha with cofactor proteins. Transfection assays using DEC1 and DEC2 mutants revealed that the C-terminal region of DEC2 is required for repression and that an LXXLL motif in DEC1 and DEC2 is necessary for RXRalpha repression. DEC1 and DEC2 repressed the induction of LXR target genes, associated with the promoter of an LXR target gene, and dissociated from the promoter with ligand treatment. Knockdown of endogenous DEC1 and DEC2 enhanced the LXR target gene expression in hepatocytes. Expression of Dec1, Dec2, and Srebp-1c showed a circadian rhythm in the liver of mice, whereas that of Lxralpha, Lxrbeta, and Rxralpha was not rhythmic. DEC1 and DEC2 also repressed the transactivation of other RXR heterodimers, such as farnesoid X receptor, vitamin D receptor, and retinoic acid receptor. Thus, the repressor function of DEC1 and DEC2 may be extended to other RXR heterodimer nuclear receptors.