Acute Promyelocytic Leukemia, Retinoic Acid, and Arsenic: A Tale of Dualities.

Acute promyelocytic leukemia (APL) is driven by the promyelocytic leukemia (PML)/retinoic acid receptor α (RARA) fusion oncoprotein. Over the years, it has emerged as a model system to understand how this simple (and sometimes sole) genetic alteration can transform hematopoietic progenitors through the acquisition of dominant-negative properties toward both transcriptional control by nuclear receptors and PML-mediated senescence. The fortuitous identification of two drugs, arsenic trioxide (ATO) and all-trans-retinoic acid (ATRA), that respectively bind PML and RARA to initiate PML/RARA degradation, has allowed an unprecedented dissection of the cellular and molecular mechanisms involved in patients' cure by the ATO/ATRA combination. This analysis has unraveled the dual and complementary roles of RARA and PML in both APL initiation and cure by the ATRA/ATO combination. We discuss how some of the features unraveled by APL studies may be more broadly applicable to some other forms of leukemia. In particular, the functional synergy between drugs that promote differentiation and those that initiate apoptosis/senescence to impede self-renewal could pave the way to novel curative combinations.

Rig1 receptor plays a critical role in cardiac reprogramming via YY1 signaling.

We discovered that innate immunity plays an important role in the reprogramming of fibroblasts into cardiomyocytes. In this report, we define the role of a novel retinoic acid-inducible gene 1 Yin Yang 1 (Rig1:YY1) pathway. We found that fibroblast to cardiomyocyte reprogramming efficacy was enhanced by specific Rig1 activators. To understand the mechanism of action, we performed various transcriptomic, nucleosome occupancy, and epigenomic approaches. Analysis of the datasets indicated that Rig1 agonists had no effect on reprogramming-induced changes in nucleosome occupancy or loss of inhibitory epigenetic motifs. Instead, Rig1 agonists were found to modulate cardiac reprogramming by promoting the binding of YY1 specifically to cardiac genes. To conclude, these results show that the Rig1:YY1 pathway plays a critical role in fibroblast to cardiomyocyte reprogramming.

A novel nuclear receptor subfamily enlightens the origin of heterodimerization.

BACKGROUND: Nuclear receptors are transcription factors of central importance in human biology and associated diseases. Much of the knowledge related to their major functions, such as ligand and DNA binding or dimerization, derives from functional studies undertaken in classical model animals. It has become evident, however, that a deeper understanding of these molecular functions requires uncovering how these characteristics originated and diversified during evolution, by looking at more species. In particular, the comprehension of how dimerization evolved from ancestral homodimers to a more sophisticated state of heterodimers has been missing, due to a too narrow phylogenetic sampling. Here, we experimentally and phylogenetically define the evolutionary trajectory of nuclear receptor dimerization by analyzing a novel NR7 subgroup, present in various metazoan groups, including cnidarians, annelids, mollusks, sea urchins, and amphioxus, but lost in vertebrates, arthropods, and nematodes. RESULTS: We focused on NR7 of the cephalochordate amphioxus B. lanceolatum. We present a complementary set of functional, structural, and evolutionary analyses that establish that NR7 lies at a pivotal point in the evolutionary trajectory from homodimerizing to heterodimerizing nuclear receptors. The crystal structure of the NR7 ligand-binding domain suggests that the isolated domain is not capable of dimerizing with the ubiquitous dimerization partner RXR. In contrast, the full-length NR7 dimerizes with RXR in a DNA-dependent manner and acts as a constitutively active receptor. The phylogenetic and sequence analyses position NR7 at a pivotal point, just between the basal class I nuclear receptors that form monomers or homodimers on DNA and the derived class II nuclear receptors that exhibit the classical DNA-independent RXR heterodimers. CONCLUSIONS: Our data suggest that NR7 represents the "missing link" in the transition between class I and class II nuclear receptors and that the DNA independency of heterodimer formation is a feature that was acquired during evolution. Our studies define a novel paradigm of nuclear receptor dimerization that evolved from DNA-dependent to DNA-independent requirements. This new concept emphasizes the importance of DNA in the dimerization of nuclear receptors, such as the glucocorticoid receptor and other members of this pharmacologically important oxosteroid receptor subfamily. Our studies further underline the importance of studying emerging model organisms for supporting cutting-edge research.

Retinoic acid receptor structures: the journey from single domains to full-length complex.

The retinoic acid receptors (RARα, ß, and γ) are multi-domain polypeptides that heterodimerize with retinoid X receptors (RXRα, ß, and γ) to form functional transcription factors. Understanding the three-dimensional molecular organization of these nuclear receptors (NRs) began with RAR and RXR DNA-binding domains (DBDs), and were followed with studies on isolated ligand-binding domains (LBDs). The more complete picture emerged in 2017 with the multi-domain crystal structure of RXRα-RARß on its response element with retinoic acid molecules and coactivator segments on both proteins. The analysis of that structure and its complementary studies have clarified the direct communication pathways within RXR-RAR polypeptides, through which DNA binding, protein-ligand, and protein-protein interactions are integrated for overall functional responses. Understanding the molecular connections in the RXR-RAR complex has benefited from direct observations of the multi-domain structures of RXRα-PPARγ, RXRα-LXRß, HNF-4α homodimer, and androgen receptor homodimer, each bound to its response element. These comprehensive NR structures show unique quaternary architectures, yet all have DBD-DBD, LBD-LBD, and DBD-LBD domain-domain contacts within them. These convergence zones allow signals from discrete domains of their polypeptides to be propagated and integrated across their entire complex, shaping their overall responses in an allosteric fashion.

Combining phenotypic profiling and targeted RNA-Seq reveals linkages between transcriptional perturbations and chemical effects on cell morphology: Retinoic acid as an example.

The United States Environmental Protection Agency has proposed a tiered testing strategy for chemical hazard evaluation based on new approach methods (NAMs). The first tier includes in vitro profiling assays applicable to many (human) cell types, such as high-throughput transcriptomics (HTTr) and high-throughput phenotypic profiling (HTPP). The goals of this study were to: (1) harmonize the seeding density of U-2 OS human osteosarcoma cells for use in both assays; (2) compare HTTr- versus HTPP-derived potency estimates for 11 mechanistically diverse chemicals; (3) identify candidate reference chemicals for monitoring assay performance in future screens; and (4) characterize the transcriptional and phenotypic changes in detail for all-trans retinoic acid (ATRA) as a model compound known for its adverse effects on osteoblast differentiation. The results of this evaluation showed that (1) HTPP conducted at low (400 cells/well) and high (3000 cells/well) seeding densities yielded comparable potency estimates and similar phenotypic profiles for the tested chemicals; (2) HTPP and HTTr resulted in comparable potency estimates for changes in cellular morphology and gene expression, respectively; (3) three test chemicals (etoposide, ATRA, dexamethasone) produced concentration-dependent effects on cellular morphology and gene expression that were consistent with known modes-of-action, demonstrating their suitability for use as reference chemicals for monitoring assay performance; and (4) ATRA produced phenotypic changes that were highly similar to other retinoic acid receptor activators (AM580, arotinoid acid) and some retinoid X receptor activators (bexarotene, methoprene acid). This phenotype was observed concurrently with autoregulation of the RARB gene. Both effects were prevented by pre-treating U-2 OS cells with pharmacological antagonists of their respective receptors. Thus, the observed phenotype could be considered characteristic of retinoic acid pathway activation in U-2 OS cells. These findings lay the groundwork for combinatorial screening of chemicals using HTTr and HTPP to generate complementary information for the first tier of a NAM-based chemical hazard evaluation strategy.

Crabp1 Modulates HPA Axis Homeostasis and Anxiety-like Behaviors by Altering FKBP5 Expression.

Retinoic acid (RA), the principal active metabolite of vitamin A, is known to be involved in stress-related disorders. However, its mechanism of action in this regard remains unclear. This study reports that, in mice, endogenous cellular RA binding protein 1 (Crabp1) is highly expressed in the hypothalamus and pituitary glands. Crabp1 knockout (CKO) mice exhibit reduced anxiety-like behaviors accompanied by a lowered stress induced-corticosterone level. Furthermore, CRH/DEX tests show an increased sensitivity (hypersensitivity) of their feedback inhibition in the hypothalamic-pituitary-adrenal (HPA) axis. Gene expression studies show reduced FKBP5 expression in CKO mice; this would decrease the suppression of glucocorticoid receptor (GR) signaling thereby enhancing their feedback inhibition, consistent with their dampened corticosterone level and anxiety-like behaviors upon stress induction. In AtT20, a pituitary gland adenoma cell line elevating or reducing Crabp1 level correspondingly increases or decreases FKBP5 expression, and its endogenous Crabp1 level is elevated by GR agonist dexamethasone or RA treatment. This study shows, for the first time, that Crabp1 regulates feedback inhibition of the the HPA axis by modulating FKBP5 expression. Furthermore, RA and stress can increase Crabp1 level, which would up-regulate FKBP5 thereby de-sensitizing feedback inhibition of HPA axis (by decreasing GR signaling) and increasing the risk of stress-related disorders.

L-carnitine exerts a nutrigenomic effect via direct modulation of nuclear receptor signaling in adipocytes, hepatocytes and SKMC, demonstrating its nutritional impact.

L-carnitine is an indispensable metabolite facilitating the transport of fatty acids into the mitochondrial matrix and has been previously postulated to exert a nutrigenomic effect. However, the underlying molecular mechanisms remain mostly unclear. We hypothesized that L-carnitine interacts with nuclear receptors involved in metabolic regulation, thereby modulating downstream targets of cellular metabolism. Therefore, we investigated the effect of L-carnitine supplementation on protein activity, mRNA expression, and binding affinities of nuclear receptors as well as mRNA expression of downstream targets in skeletal muscle cells, hepatocytes, and differentiated adipocytes. L-carnitine supplementation to hepatocytes increased the protein activity of multiple nuclear receptors (RAR, RXR, VDR, PPAR, HNF4, ER, LXR). Diverging effects on the mRNA expression of PPAR-α, PPAR-δ, PPAR-γ, RAR-ß, LXR-α, and RXR-α were observed in adipocytes, hepatocytes, and skeletal muscle cells. mRNA levels of PPAR-α, a key regulator of lipolysis and ß-oxidation, were significantly upregulated, emphasizing a role of L-carnitine as a promoter of lipid catabolism. L-carnitine administration to hepatocytes modulated the transcription of key nuclear receptor target genes, including ALDH1A1, a promoter of adipogenesis, and OGT, a contributor to insulin resistance. Electrophoretic mobility shift assays proved L-carnitine to increase binding affinities of nuclear receptors to their promoter target sequences, suggesting a molecular mechanism for the observed transcriptional modulation. Overall, these findings indicate that L-carnitine modulates the activity and expression of nuclear receptors, thereby promoting lipolytic gene expression and decreasing transcription of target genes linked to adipogenesis and insulin resistance.

DNA recognition by retinoic acid nuclear receptors.

Retinoic acid receptors (RARs) heterodimerize with retinoid X receptors (RXRs) to regulate gene expression. The heterodimer recognizes the genome via a large and diverse repertoire of DNA response elements. Assessing the binding mode of RAR and RXR with various DNA response elements is important for understanding how they select their binding site and how DNA sequence and topology allosterically regulate RAR function. A number of complementary assays are often employed for analysis of the binding mode. To biochemically and structurally characterize RAR and RXR-DNA complexes, we describe how to express and purify RAR and RXR-DNA binding domains (DBDs) and multidomain constructs. We also describe the use of electrospray ionization mass spectrometry (ESI MS) and isothermal titration calorimetry (ITC) that give information about stoichiometry and binding affinity, as well as our approaches for co-crystallization of RAR and RXR DBDs with DNA.

Apo-14´-Carotenoic Acid Is a Novel Endogenous and Bioactive Apo-Carotenoid.

Carotenoids can be metabolized to various apo-carotenoids and retinoids. Apo-15´-carotenoic acid (retinoic acid, RA) is a potent activator of the retinoic acid receptor (RAR) in its all-trans- (ATRA) and 9-cis- (9CRA) forms. In this study we show firstly, that apo-14´-carotenoic acid (A14CA), besides retinoic acids, is present endogenously and with increased levels in the human organism after carrot juice supplementation rich in ß-carotene. All-trans-A14C (ATA14CA) is just a moderate activator of RAR-transactivation in reporter cell lines but can potently activate retinoic acid response element (RARE)-mediated signalling in DR5/RARE-reporter mice and potently increase retinoid-reporter target gene expression in ATA14CA-supplemented mice and treated MM6 cells. Further metabolism to all-trans-13,14-dihydroretinoic acid (ATDHRA) may be the key for its potent effects on retinoid target gene activation in ATA14CA-treated MM6 cells and in liver of supplemented mice. We conclude that besides RAs, there are alternative ways to activate RAR-response pathways in the mammalian organism. ATA14CA alone and in combination with its metabolite ATDHRA may be an alternative pathway for potent RAR-mediated signalling.

Correlating blood-based DNA methylation markers and prostate cancer risk in African-American men.

The objective of this work was to investigate the clinical significance of promoter gene DNA methylation changes in whole blood from African-American (AA) men with prostate cancer (PCa). We used high throughput pyrosequencing analysis to quantify percentage DNA methylation levels in a panel of 8 genes (RARß2, TIMP3, SPARC, CDH13, HIN1, LINE1, CYB5R2 and DRD2) in blood DNA obtained from PCa and non-cancerous controls cases. Correlations of methylation status and various clinicopathological features were evaluated. Six genes tested achieved significant difference in DNA methylation levels between the PCa compared to control cases (P < 0.05). The TIMP3 loci demonstrated significant correlation of DNA methylation with age for all cases analyzed (p < 0.05). We observed an inverse correlation between CDH13 methylation (p = 0.045; r = -0.21) and serum vitamin D level whereas TIMP3 methylation (p = 0.021; r = -0.24) and DRD2 methylation (p = 0.056; r = -0.201) showed inverse correlation with supplementary vitamin D in the cancer cases. We also observed a direct correlation between methylation of RARß2 (p = 0.0036; r = 0.293) and SPARC (p = 0.0134; r = 0.20) loci with PSA level in the controls but not the cancer cases. In addition, alcohol cases significantly correlated with higher RARß2 methylation (p = 0.0314) in comparison with non-alcohol cases. Furthermore, we observed an inverse correlation of DRD2 methylation (p = 0.0349; r = -0.343) and Gleason score. Our data suggests that promoter methylation occurred more frequently in the blood of AA PCa and is associated with various clinicopathological features in AA men with PCa.

Identification of Retinoic Acid Receptor Agonists as Potent Hepatitis B Virus Inhibitors via a Drug Repurposing Screen.

Currently available therapies for chronic hepatitis B virus (HBV) infection can efficiently reduce viremia but induce hepatitis B surface antigen (HBsAg) loss in very few patients; also, these therapies do not greatly affect the viral covalently closed circular DNA (cccDNA). To discover new agents with complementary anti-HBV effects, we performed a drug repurposing screen of 1,018 Food and Drug Administration (FDA)-approved compounds using HBV-infected primary human hepatocytes (PHH). Several compounds belonging to the family of retinoic acid receptor (RAR) agonists were identified that reduced HBsAg levels in a dose-dependent manner without significant cytotoxicity. Among them, tazarotene exhibited the most potent anti-HBV effect, with a half-maximal inhibitory concentration (IC50) for HBsAg of less than 30 nM in PHH. The inhibitory effect was also observed in HBV-infected differentiated HepaRG (dHepaRG) models, but not in HepG2.215 cells, and HBV genotypes A to D were similarly inhibited. Tazarotene was further demonstrated to repress HBV cccDNA transcription, as determined by the levels of HBV cccDNA and RNAs and the activation of HBV promoters. Moreover, RNA sequence analysis showed that tazarotene did not induce an interferon response but altered the expression of a number of genes associated with RAR and metabolic pathways. Inhibition of RARß, but not RARα, by a specific antagonist significantly attenuated the anti-HBV activity of tazarotene, suggesting that tazarotene inhibits HBV in part through RARß. Finally, a synergistic effect of tazarotene and entecavir on HBV DNA levels was observed. Therefore, RAR agonists as represented by tazarotene were identified as potential novel anti-HBV agents.

Supplementation of all trans retinoic acid ameliorates ethanol-induced endoplasmic reticulum stress.

CONTEXT: Molecular pathogenesis of chronic alcoholism is linked to increased endoplasmic reticulum stress. Ethanol is a competitive inhibitor of vitamin A metabolism and vitamin A supplementation aggravates existing liver problems. Hence, we probed into the impact of supplementation of all trans retinoic acid (ATRA), the active metabolite of vitamin A on ethanol-induced endoplasmic reticulcum stress. METHODS: Male Sprague-Dawley rats were divided into four groups - I: Control; II: Ethanol; III: ATRA; IV: ATRA + Ethanol. After 90 days the animals were sacrificed to study markers of lipid peroxidation in hepatic microsomal fraction and expression of ER stress proteins and apoptosis in liver. RESULTS AND CONCLUSION: Ethanol caused hepatic hyperlipidemia, enhanced microsomal lipid peroxidation, upregulated expression of unfolded protein response associated proteins and that of apoptosis. Ethanol also led to downregulation of retinoid receptors. ATRA supplementation reversed all these alterations indicating the decrease in ethanol-induced endoplasmic reticulum stress.

Retinoic Acid Facilitates Toll-Like Receptor 4 Expression to Improve Intestinal Barrier Function through Retinoic Acid Receptor Beta.

BACKGROUND/AIMS: Vitamin A (VA) protects the intestinal epithelial barrier by improving cell migration and proliferation. Our previous studies demonstrated that VA deficiency (VAD) during pregnancy suppresses the systemic and mucosal immune responses in the intestines of offspring and that VA supplementation (VAS) during early life can increase immune cell counts. However, little is known about the mechanisms by which VA regulates intestinal epithelial barrier function. METHODS: Caco-2 cells were treated with all-trans retinoic acid (ATRA) for 24 hours to determine the optimum ATRA concentration to which the cells in question respond. Caco-2 cells were infected with recombinant adenoviruses carrying retinoic acid receptor beta (Ad-RARß) and small interfering RARß(siRARß) to assess the effects of RARß signalling on the expression of specific proteins. A siTLR4 lentivirus was used to knockdown Toll-like receptor 4 (TLR4) in Caco-2 cells to determine its role in the protective effects of VA on the intestinal epithelial barrier, and experiments involving TLR4-knock-out mice were performed to verify the effect of TLR4. VA normal (VAN), VAD and VAS rat models were established to confirm that changes in RARß, TLR4 and ZO-2 expression levels that occurred following decreases or increases in retinol concentrations in vivo, and the permeability of the Caco-2 cell monolayer, as well as that of the epithelial barrier of the rat intestine was detected by measuring transepithelial resistance (TER) or performing enzyme-linked immunosorbent assay (ELISA). Retinoic acid receptor (RAR), toll like receptor (TLR) and tight junction (TJ) mRNA and protein expression levels in Caco-2 cells and the colon monolayers in the rat and mouse models were measured by PCR and western blotting, respectively. Co-immunoprecipitation (co-IP) and immunofluorescence staining were performed to assess the interactions among RARß, TLR4 and zonula occluden-2 (ZO-2) in Caco-2 cells, and chromatin immunoprecipitation (ChIP) assay was performed to assess the binding between RARß and the TLR4 promoter sequence in Caco-2 cells. RESULTS: In the present study, ATRA treatment not only increased the TER of the Caco-2 monolayer but also up-regulated the expression levels of RARß, TLR4 and ZO-2 in Caco-2 cells. The expression levels of these three proteins were significantly decreased in the colonic epithelial monolayers of VAD rats compared with those of VAN rats and were significantly increased following VAS in the corresponding group compared with the control group. Furthermore, the above changes in TLR4 and ZO-2 expression levels were augmented or attenuated by Ad-RARß or siRARß infection, respectively, in Caco-2 cells. Interestingly, siTLR4 down-regulated ZO-2 expression but did not affect RARß expression in Caco-2 cells, and in VAD mice the lack of TLR4 did not affect ZO-2 expression. We noted direct interactions between RARß and TLR4, TLR4 and ZO-2 in Caco-2 cells, and ChIP assay showed that RARß could bind to the TLR4 promoter but not the ZO-2 promoter in Caco-2 cells. CONCLUSION: Taken together, our results indicate that RARß enhanced ZO-2 expression by regulating TLR4 to improve intestinal epithelial barrier function in Caco-2 cells, as well as in rat and mouse models, but not in humans.

Genetic and pharmacological inhibition of retinoic acid receptor γ function promotes endochondral bone formation.

The nuclear retinoic acid receptors (RARs) play key roles in skeletal development and endochondral ossification. Previously, we showed that RARγ regulates chondrogenesis and that pharmacological activation of RARγ blocked heterotopic ossification (HO), pathology in which endochondral bone forms in soft tissues. Thus, we reasoned that pharmacological inhibition of RARγ should enhance endochondral ossification, leading to a potential therapeutic strategy for bone deficiencies. We created surgical bone defects in wild type and RARγ-null mice and monitored bone healing. Fibrous, cartilaginous, and osseous tissues formed in both groups by day 7, but more cartilaginous tissue formed in mutants within and around the defects compared to controls. Next, we implanted a mixture of Matrigel and rhBMP2 subdermally to induce ectopic endochondral ossification. Administration of RARγ antagonists significantly stimulated ectopic bone formation in wild type but not in RARγ-null mice. The antagonist-induced increases in bone formation were preceded by increases in cartilage formation and were accompanied by higher levels of phosphorylated Smad1/5/8 (pSmad1/5/8) compared to vehicle-treated control. Higher pSmad1/5/8 levels were also observed in cartilaginous tissues forming in healing bone defects in RARγ-null mice, and increases in pSmad1/5/8 levels and Id1-luc activity were observed in RARγ antagonist-treated chondrogenic cells in culture. Our data show that genetic or pharmacological interference with RARγ stimulates endochondral bone formation and does so at least in part by stimulating canonical BMP signaling. This pharmacologic strategy could represent a new tool to enhance endochondral bone formation in the setting of various orthopedic surgical interventions and other skeletal deficiencies. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1096-1105, 2017.

Reduced adiponectin expression after high-fat diet is associated with selective up-regulation of ALDH1A1 and further retinoic acid receptor signaling in adipose tissue.

Adiponectin is an adipocyte-derived adipokine with potent antidiabetic, anti-inflammatory, and antiatherogenic activity. Long-term, high-fat diet results in gain of body weight, adiposity, further inflammatory-based cardiovascular diseases, and reduced adiponectin secretion. Vitamin A derivatives/retinoids are involved in several of these processes, which mainly take place in white adipose tissue (WAT). In this study, we examined adiponectin expression as a function of dietary high-fat and high-vitamin A conditions in mice. A decrease of adiponectin expression in addition to an up-regulation of aldehyde dehydrogenase A1 (ALDH1A1), retinoid signaling, and retinoic acid response element signaling was selectively observed in WAT of mice fed a normal-vitamin A, high-fat diet. Reduced adiponectin expression in WAT was also observed in mice fed a high-vitamin A diet. Adipocyte cell culture revealed that endogenous and synthetic retinoic acid receptor (RAR)α- and RARγ-selective agonists, as well as a synthetic retinoid X receptor agonist, efficiently reduced adiponectin expression, whereas ALDH1A1 expression only increased with RAR agonists. We conclude that reduced adiponectin expression under high-fat dietary conditions is dependent on 1) increased ALDH1A1 expression in adipocytes, which does not increase all-trans-retinoic acid levels; 2) further RAR ligand-induced, WAT-selective, increased retinoic acid response element-mediated signaling; and 3) RAR ligand-dependent reduction of adiponectin expression.-Landrier, J.-F., Kasiri, E., Karkeni, E., Mihály, J., Béke, G., Weiss, K., Lucas, R., Aydemir, G., Salles, J., Walrand, S., de Lera, A. R., Rühl, R. Reduced adiponectin expression after high-fat diet is associated with selective up-regulation of ALDH1A1 and further retinoic acid receptor signaling in adipose tissue.

Lycopene supplementation restores vitamin A deficiency in mice and possesses thereby partial pro-vitamin A activity transmitted via RAR signaling.

SCOPE: The aim of this study was to compare if lycopene also possesses pro-vitamin A (VA) activity comparable to known VA derivatives. MATERIALS AND METHODS: We used a transgenic retinoic acid response element reporter mouse model (n = 8, per group) for this study, and after the initial wash out of VA using a vitamin A deficient diet (VAD) for 18 weeks, the animals were supplemented further with (a) VAD-fed mice, (b) VAD-fed mice plus retinol (20 mg/kg bw), (c) VAD-fed mice plus ß-carotene (40 mg/kg bw), and (d) VAD-fed mice plus lycopene (40 mg/kg bw). Using ex vivo scanning and gene expression analysis of retinoid target and VA marker gene analysis in various organs of these supplemented mice (b, c, d), we found increased luciferase activity and normalized marker and target gene analysis compared to group a. CONCLUSIONS: Lycopene can restore VA deficiency and compensate VA for retinoic acid receptor (RAR)-mediated signaling as the major function of VA in the mammalian organism. Lycopene administration can initiate upregulation of RAR-mediated signaling in various organs in VAD-fed animals via potential novel bioactive lycopene metabolites. This indicates that lycopene possesses partial pro-VA activity in mice transmitted via RAR-mediated signaling.

Association between nuclear expression of retinoic acid receptor alpha and beta and clinicopathological features and prognosis of advanced non-small cell lung cancer.

BACKGROUND: Transcription factors such as retinoic acid receptor alpha (RARα) and beta (RARß) and Yin Yang 1 (YY1) are associated with the progression of non-small cell lung cancer (NSCLC). In particular, a lack of RARß expression is associated with NSCLC development. The aim of this study was to analyze the expression of RARα, RARß and YY1 and their relationship with prognosis in patients with advanced NSCLC. METHODS: The expression of RARα, RARß and YY1 was assessed by immunohistochemistry and quantitative computerized image software. RESULTS: Eighty-five patients treated with platinum-based chemotherapy were included in the analysis. The mean and standard deviation of the nuclear expression of RARα, RARß and YY1 were 184.5 ± 124.4, 18 ± 27 and 16.6 ± 20.5, respectively. The nuclear expression of RARß was associated with the nuclear expression of YY1 (R 2 = 0.28; p value < 0.0001). Patients with high nuclear expression of YY1 were likely to be non-smokers (61.9 vs 40.5 %). Median progression-free survival (PFS) was 5.9 months (3.48-8.28). Low expression of RARα was independently associated with worse PFS following chemotherapy (10.3 vs 5.46 months p = 0.040). Median overall survival (OS) was 15.6 months (4.5-26.7), and lower nuclear expression of RARß was independently associated with shorter OS (27.5 vs 8.7 months; p = 0.037). CONCLUSION: Our study suggests that the loss of RARs is associated with a worse prognosis and these receptors could be a potential molecular target for NSCLC.

The antitumor effect of TIG3 in liver cancer cells is involved in ERK1/2 inhibition.

Tazarotene-induced gene 3 (TIG3) was first characterized in tazarotene-treated human keratinocytes and identified as a retinoic acid responder gene, an important mediator of antitumor effects by retinoids. In this study, we aim to investigate the inhibitory effect of TIG3 on the growth of liver cancer and explore its underlying mechanism. Human hepatocellular carcinoma (HCC) Hep3B cells were transfected with plasmid GV141 carrying full-length TIG3 complementary DNA (cDNA). The effects of TIG3 on cell proliferation, apoptosis, and migration were determined in vitro. The suppressor effect of TIG3 on tumor growth was evaluated in vivo in a nude mouse HCC model. We observed that TIG3 expression is decreased in the Hep3B cell line as well as primary HCC tumors, and TIG3 expression inversely correlates with Ki-67 expression. Overexpression of TIG3 suppresses tumor growth in HCC both in vitro and in vivo via ERK1/2 inhibition by promoting apoptosis and inhibiting proliferation and migration. These findings identify TIG3 as an attractive therapeutic target for HCC.

Family of microRNA-146 Regulates RARß in Papillary Thyroid Carcinoma.

Retinoic acid is a promising tool in adjuvant cancer therapies, including refractory thyroid cancer, and its biological role is mediated by the retinoic acid receptor beta (RARß). However, expression of RARß is lowered in papillary thyroid carcinoma (PTC), contributing to promotion of tumor growth and inefficiency of retinoic acid and radioactive iodine treatment. The causes of aberrant RARB expression are largely unknown. We hypothesized that the culpable mechanisms include the action of microRNAs from the miR-146 family, previously identified as significantly upregulated in PTC tumors. To test this hypothesis, we assessed the expression of RARB as well as miR-146a-5p and miR-146b-5p in 48 PTC tumor/normal tissue pairs by Taqman assay to reveal that the expression of RARB was 3.28-fold decreased, and miR-146b-5p was 28.9-fold increased in PTC tumors. Direct interaction between miRs and RARB was determined in the luciferase assay and further confirmed in cell lines, where overexpression of miR-146a-5p and miR-146b-5p caused a 31% and 33% decrease in endogenous RARB mRNA levels. Inhibition of miR-146a and miR-146b resulted in 62.5% and 45.4% increase of RARB, respectively, and a concomitant decrease in proliferation rates of thyroid cancer cell lines, analyzed in xCELLigence system.We showed that two microRNAs of the miR-146 family directly regulate RARB. Inhibition of miRs resulted in restoration of RARB expression and decreased rates of proliferation of thyroid cancer cells. By restoring RARB levels, microRNA inhibitors may become part of an adjuvant therapy in thyroid cancer patients.

Effects of conjugated linoleic acid and lutein on the growth performance and immune response of broiler chickens.

The effects of lutein and conjugated linoleic acid (CLA) on growth performance and immune response of broiler chickens were evaluated in the presence and absence of Salmonella lipopolysaccharide (LPS) immune challenge. Cobb chicks (360; 1 to 22 d of age) were used in a 3 × 2 factorial arrangement of CLA (0, 1, and 2%) and lutein (0 and 50 mg/kg) dietary levels. At d 8 and 15, birds were injected with BSA to assess IgY production. At d 20, birds were injected with LPS. Samples of liver, spleen, and duodenum were collected at 3 and 16 h post-LPS challenge for RT-qPCR analysis of RXRα, RXRγ, PPARα, PPARγ, TLR-4, IL-1ß, IL-2, IL-10, and IL-12 gene expression. CLA decreased BW, BW gain (BWG), and G:F from d 1 to 20, but these effects were reversed when lutein was included in the 1% CLA diet (P < 0.001). The production of IgY anti-BSA increased following a 2% CLA supplementation (P < 0.01). LPS increased the liver:BW ratio at 3 h post-injection (P < 0.001) and decreased BWG at 3, 16, and 40 h (P < 0.001). Lutein decreased plasmatic nitric oxide levels (P < 0.01). LPS downregulated PPARα mRNA in the duodenum (P = 0.02) and liver (P = 0.04), and PPARγ (P = 0.01) and RXRα (P = 0.08) in the spleen; these effects were not reversed by CLA or lutein as initially hypothesized. Although LPS upregulated IL-1ß (P = 0.02) and IL-12 (P = 0.07) expression, lutein downregulated these pro-inflammatory cytokines in the liver (P = 0.03 and P = 0.07, respectively). Lutein decreased splenic (P = 0.09) but increased hepatic (P = 0.06) TLR-4 mRNA. A dietary CLA supplementation of 2% increased hepatic RXRα (P = 0.10). In conclusion, CLA decreased broiler chicken growth performance, but lutein could prevent this negative effect (depending on CLA dose). Lutein had an anti-inflammatory effect, and a 2% CLA supplementation improved the humoral immune response.