The Endogenous Retinoic Acid Receptor Pathway Is Exploited by Mycobacterium tuberculosis during Infection, Both In Vitro and In Vivo.

Retinoic acid (RA) is a fundamental vitamin A metabolite involved in regulating immune responses through the nuclear RA receptor (RAR) and retinoid X receptor. While performing experiments using THP-1 cells as a model for Mycobacterium tuberculosis infection, we observed that serum-supplemented cultures displayed high levels of baseline RAR activation in the presence of live, but not heat-killed, bacteria, suggesting that M. tuberculosis robustly induces the endogenous RAR pathway. Using in vitro and in vivo models, we have further explored the role of endogenous RAR activity in M. tuberculosis infection through pharmacological inhibition of RARs. We found that M. tuberculosis induces classical RA response element genes such as CD38 and DHRS3 in both THP-1 cells and human primary CD14+ monocytes via a RAR-dependent pathway. M. tuberculosis-stimulated RAR activation was observed with conditioned media and required nonproteinaceous factor(s) present in FBS. Importantly, RAR blockade by (4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphthalen-2-yl]ethenyl]benzoic acid), a specific pan-RAR inverse agonist, in a low-dose murine model of tuberculosis significantly reduced SIGLEC-F+CD64+CD11c+high alveolar macrophages in the lungs, which correlated with 2× reduction in tissue mycobacterial burden. These results suggest that the endogenous RAR activation axis contributes to M. tuberculosis infection both in vitro and in vivo and reveal an opportunity for further investigation of new antituberculosis therapies.

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.

Actin cytoskeleton remodeling primes RIG-I-like receptor activation.

The current dogma of RNA-mediated innate immunity is that sensing of immunostimulatory RNA ligands is sufficient for the activation of intracellular sensors and induction of interferon (IFN) responses. Here, we report that actin cytoskeleton disturbance primes RIG-I-like receptor (RLR) activation. Actin cytoskeleton rearrangement induced by virus infection or commonly used reagents to intracellularly deliver RNA triggers the relocalization of PPP1R12C, a regulatory subunit of the protein phosphatase-1 (PP1), from filamentous actin to cytoplasmic RLRs. This allows dephosphorylation-mediated RLR priming and, together with the RNA agonist, induces effective RLR downstream signaling. Genetic ablation of PPP1R12C impairs antiviral responses and enhances susceptibility to infection with several RNA viruses including SARS-CoV-2, influenza virus, picornavirus, and vesicular stomatitis virus. Our work identifies actin cytoskeleton disturbance as a priming signal for RLR-mediated innate immunity, which may open avenues for antiviral or adjuvant design.

Feedback between a retinoid-related nuclear receptor and the let-7 microRNAs controls the pace and number of molting cycles in C. elegans.

Animal development requires coordination among cyclic processes, sequential cell fate specifications, and once-a-lifetime morphogenic events, but the underlying timing mechanisms are not well understood. Caenorhabditis elegans undergoes four molts at regular 8 to 10 hour intervals. The pace of the cycle is governed by PERIOD/lin-42 and other as-yet unknown factors. Cessation of the cycle in young adults is controlled by the let-7 family of microRNAs and downstream transcription factors in the heterochronic pathway. Here, we characterize a negative feedback loop between NHR-23, the worm homolog of mammalian retinoid-related orphan receptors (RORs), and the let-7 family of microRNAs that regulates both the frequency and finite number of molts. The molting cycle is decelerated in nhr-23 knockdowns and accelerated in let-7(-) mutants, but timed similarly in let-7(-) nhr-23(-) double mutants and wild-type animals. NHR-23 binds response elements (ROREs) in the let-7 promoter and activates transcription. In turn, let-7 dampens nhr-23 expression across development via a complementary let-7-binding site (LCS) in the nhr-23 3' UTR. The molecular interactions between NHR-23 and let-7 hold true for other let-7 family microRNAs. Either derepression of nhr-23 transcripts by LCS deletion or high gene dosage of nhr-23 leads to protracted behavioral quiescence and extra molts in adults. NHR-23 and let-7 also coregulate scores of genes required for execution of the molts, including lin-42. In addition, ROREs and LCSs isolated from mammalian ROR and let-7 genes function in C. elegans, suggesting conservation of this feedback mechanism. We propose that this feedback loop unites the molting timer and the heterochronic gene regulatory network, possibly by functioning as a cycle counter.

Revisiting APP secretases: an overview on the holistic effects of retinoic acid receptor stimulation in APP processing.

Alzheimer's disease (AD) is the leading cause of dementia worldwide and is characterized by the accumulation of the ß-amyloid peptide (Aß) in the brain, along with profound alterations in phosphorylation-related events and regulatory pathways. The production of the neurotoxic Aß peptide via amyloid precursor protein (APP) proteolysis is a crucial step in AD development. APP is highly expressed in the brain and is complexly metabolized by a series of sequential secretases, commonly denoted the α-, ß-, and γ-cleavages. The toxicity of resulting fragments is a direct consequence of the first cleaving event. ß-secretase (BACE1) induces amyloidogenic cleavages, while α-secretases (ADAM10 and ADAM17) result in less pathological peptides. Hence this first cleavage event is a prime therapeutic target for preventing or reverting initial biochemical events involved in AD. The subsequent cleavage by γ-secretase has a reduced impact on Aß formation but affects the peptides' aggregating capacity. An array of therapeutic strategies are being explored, among them targeting Retinoic Acid (RA) signalling, which has long been associated with neuronal health. Additionally, several studies have described altered RA levels in AD patients, reinforcing RA Receptor (RAR) signalling as a promising therapeutic strategy. In this review we provide a holistic approach focussing on the effects of isoform-specific RAR modulation with respect to APP secretases and discuss its advantages and drawbacks in subcellular AD related events.

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.

Dietary Supplementation with Biobran/MGN-3 Increases Innate Resistance and Reduces the Incidence of Influenza-like Illnesses in Elderly Subjects: A Randomized, Double-Blind, Placebo-Controlled Pilot Clinical Trial.

Influenza-like illness (ILI) remains a major cause of severe mortality and morbidity in the elderly. Aging is associated with a decreased ability to sense pathogens and mount effective innate and adaptive immune responses, thus mandating the development of protective nutraceuticals. Biobran/MGN-3, an arabinoxylan from rice bran, has potent anti-aging and immunomodulatory effects, suggesting that it may be effective against ILI. The objective of the current study was to investigate the effect of Biobran/MGN-3 on ILI incidence, natural killer (NK) cell activity, and the expressions of RIG-1 (retinoic acid-inducible gene 1), MDA5 (melanoma differentiation-associated protein 5), and their downstream signaling genes ISG-15 (interferon-stimulated genes 15) and MX1 (myxovirus (influenza) resistance 1, interferon-inducible). A double-blind, placebo-controlled clinical trial included eighty healthy older adults over 55 years old, 40 males and 40 females, who received either a placebo or Biobran/MGN-3 (500 mg/day) for 3 months during known ILI seasonality (peak incidence) in Egypt. The incidence of ILI was confirmed clinically according to the WHO case definition criteria. Hematological, hepatic, and renal parameters were assessed in all subjects, while the activity of NK and NKT (natural killer T) cells was assessed in six randomly chosen subjects in each group by the degranulation assay. The effect of Biobran/MGN-3 on RIG-1 and MDA5, as well as downstream ISG15 and MX1, was assessed in BEAS-2B pulmonary epithelial cells using flow cytometry. The incidence rate and incidence density of ILI in the Biobran/MGN-3 group were 5.0% and 0.57 cases per 1000 person-days, respectively, compared to 22.5% and 2.95 cases per 1000 person-days in the placebo group. Furthermore, Biobran/MGN-3 ingestion significantly enhanced NK activity compared to the basal levels and to the placebo group. In addition, Biobran/MGN-3 significantly upregulated the expression levels of RIG-1, MDA5, ISG15, and MX1 in the human pulmonary epithelial BEAS-2B cell lines. No side effects were observed. Taken together, Biobran/MGN-3 supplementation enhanced the innate immune response of elderly subjects by upregulating the NK activity associated with reduction of ILI incidence. It also upregulated the intracellular RIG-1, MDA5, ISG15, and MX1 expression in pulmonary epithelial tissue cultures. Biobran/MGN-3 could be a novel agent with prophylactic effects against a wide spectrum of respiratory viral infections that warrants further investigation.

Disparate roles of retinoid acid signaling molecules in kidney disease.

Retinoid acid (RA) is synthesized mainly in the liver and has multiple functions in development, cell differentiation and proliferation, and regulation of inflammation. RA has been used to treat multiple diseases, such as cancer and skin disorders. The kidney is a major organ for RA metabolism, which is altered in the diseased condition. RA is known to have renal-protective effects in multiple animal models of kidney disease. RA has been shown to ameliorate podocyte injury through induction of expression of differentiation markers and regeneration of podocytes from its progenitor cells in animal models of kidney disease. The effects of RA in podocytes are mediated mainly by activation of the cAMP/PKA pathway via RA receptor-α (RARα) and activation of its downstream transcription factor, Kruppel-like factor 15. Screening of RA signaling molecules in human kidney disease has revealed RAR responder protein 1 (RARRES1) as a risk gene for glomerular disease progression. RARRES1, a podocyte-specific growth arrest gene, is regulated by high doses of both RA and TNF-α. Mechanistically, RARRES1 is cleaved by matrix metalloproteinases to generate soluble RARRES1, which then induces podocyte apoptosis through interaction with intracellular RIO kinase 1. Therefore, a high dose of RA may induce podocyte toxicity through upregulation of RARRES1. Based on the current findings, to avoid potential side effects, we propose three strategies to develop future therapies of RA for glomerular disease: 1) develop RARα- and Kruppel-like factor 15-specific agonists, 2) use the combination of a low dose of RAR-α agonist with phosphodiesterase 4 inhibitors, and 3) use a combination of RARα agonist with RARRES1 inhibitors.NEW & NOTEWORTHY Retinoic acid (RA) exerts pleotropic cellular effects, including induction of cell differentiation while inhibiting proliferation and inflammation. These effects are mediated by both RA responsive element-dependent or -independent pathways. In kidneys, RA confers renoprotection by signaling through podocyte RA receptor (RAR)α and activation of cAMP/PKA/Kruppel-like factor 15 pathway to promote podocyte differentiation. Nevertheless, in kidney disease settings, RA can also promote podocyte apoptosis and loss through downstream expression of RAR responder protein 1, a recently described risk factor for glomerular disease progression. These disparate roles of RA underscore the complexity of its effects in kidney homeostasis and disease, and a need to target specific RA-mediated pathways for effective therapeutic treatments against kidney disease progression.

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.

Epithelium intrinsic vitamin A signaling co-ordinates pathogen clearance in the gut via IL-18.

Intestinal epithelial cells (IECs) are at the forefront of host-pathogen interactions, coordinating a cascade of immune responses to protect against pathogens. Here we show that IEC-intrinsic vitamin A signaling restricts pathogen invasion early in the infection and subsequently activates immune cells to promote pathogen clearance. Mice blocked for retinoic acid receptor (RAR) signaling selectively in IECs (stopΔIEC) showed higher Salmonella burden in colonic tissues early in the infection that associated with higher luminal and systemic loads of the pathogen at later stages. Higher pathogen burden in stopΔIEC mice correlated with attenuated mucosal interferon gamma (IFNγ) production by underlying immune cells. We found that, at homeostasis, the intestinal epithelium of stopΔIEC mice produced significantly lower amounts of interleukin 18 (IL-18), a potent inducer of IFNγ. Regulation of IL-18 by vitamin A was also observed in a dietary model of vitamin A supplementation. IL-18 reconstitution in stopΔIEC mice restored resistance to Salmonella by promoting epithelial cell shedding to eliminate infected cells and limit pathogen invasion early in infection. Further, IL-18 augmented IFNγ production by underlying immune cells to restrict pathogen burden and systemic spread. Our work uncovers a critical role for vitamin A in coordinating a biphasic immune response to Salmonella infection by regulating IL-18 production by IECs.

The Roles of GSK-3ß in Regulation of Retinoid Signaling and Sorafenib Treatment Response in Hepatocellular Carcinoma.

Rationale: Glycogen synthase kinase-3ß (GSK-3ß) plays key roles in metabolism and many cellular processes. It was recently demonstrated that overexpression of GSK-3ß can confer tumor growth. However, the expression and function of GSK-3ß in hepatocellular carcinoma (HCC) remain largely unexplored. This study is aimed at investigating the role and therapeutic target value of GSK-3ß in HCC. Methods: We firstly clarified the expression of GSK-3ß in human HCC samples. Given that deviated retinoid signalling is critical for HCC development, we studied whether GSK-3ß could be involved in the regulation. Since sorafenib is currently used to treat HCC, the involvement of GSK-3ß in sorafenib treatment response was determined. Co-immunoprecipitation, GST pull down, in vitro kinase assay, luciferase reporter and chromatin immunoprecipitation were used to explore the molecular mechanism. The biological readouts were examined with MTT, flow cytometry and animal experiments. Results: We demonstrated that GSK-3ß is highly expressed in HCC and associated with shorter overall survival (OS). Overexpression of GSK-3ß confers HCC cell colony formation and xenograft tumor growth. Tumor-associated GSK-3ß is correlated with reduced expression of retinoic acid receptor-ß (RARß), which is caused by GSK-3ß-mediated phosphorylation and heterodimerization abrogation of retinoid X receptor (RXRα) with RARα on RARß promoter. Overexpression of functional GSK-3ß impairs retinoid response and represses sorafenib anti-HCC effect. Inactivation of GSK-3ß by tideglusib can potentiate 9-cis-RA enhancement of sorafenib sensitivity (tumor inhibition from 48.3% to 93.4%). Efficient induction of RARß by tideglusib/9-cis-RA is required for enhanced therapeutic outcome of sorafenib, which effect is greatly inhibited by knocking down RARß. Conclusions: Our findings demonstrate that GSK-3ß is a disruptor of retinoid signalling and a new resistant factor of sorafenib in HCC. Targeting GSK-3ß may be a promising strategy for HCC treatment in clinic.

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.

Discovery and lead optimisation of a potent, selective and orally bioavailable RARß agonist for the potential treatment of nerve injury.

Oxadiazole replacement of an amide linkage in an RARα agonist template 1, followed by lead optimisation, has produced a highly potent and selective RARß agonist 4-(5-(4,7-dimethylbenzofuran-2-yl)-1,2,4-oxadiazol-3-yl)benzoic acid (10) with good oral bioavailability in the rat and dog. This molecule increases neurite outgrowth in vitro and induces sensory axon regrowth in vivo in a rodent model of avulsion and crush injury, and thus has the potential for the treatment of nerve injury.

Beta-carotene as a novel therapy for the treatment of "Autistic like behavior" in animal models of Autism.

Autism-affected individuals are characterized by lower plasma oxytocin and its ectoenzyme regulator CD38. Oxytocin, a hypothalamic hormone secreted upon the release of CD38, plays a role in social behavior and bonding. All-trans retinoic acid is a potent inducer of CD38 and can be used as a novel therapeutic strategy in autism. We investigated the role of beta-carotene in rescuing autistic-like behavior in BALB/c and BTBR mice. Beta-carotene derivatives are preferred as they are neither toxic nor teratogenic. Beta-carotene at 0.1-5.0 mg/kg was administered orally to BALB/c and BTBR newborn mice on days 1-7. They were tested at age 2-3 months for five behavioral tests for "autism"; in addition, brain CD38, oxytocin, oxytocin receptor, Brain Derived Neurotrophic Factor (BDNF) and retinoic acid receptor gene expression, serum oxytocin levels, and neurological score were evaluated. Beta-carotene administered at birth significantly increased T-maze alternations and led to longer time spent with an unfamiliar mouse in the "three-chamber test" and less time spent in the empty chamber. Furthermore, enhanced activity in the open field test; increased time spent in the reciprocal social interaction test; decreased grooming and bedding behaviors; and enhanced brain CD38, oxytocin, oxytocin receptor, BDNF, retinoic acid gene expression, and serum oxytocin levels. No changes in neurological score were observed. Beta-carotene oral supplementation to BALB/c and BTBR mice at birth significantly reduced restricted and stereotyped behaviors and interests, increased social interactions and communication, CD38, and oxytocin, probably by enhancing brain neuroplasticity without toxicity. Thus, beta-carotene administered after birth to newborns of families predisposed to "autism" has the potential to prevent/ameliorate" autistic like behavior". These results support further clinical studies.

Molecular mechanism and cytotoxicity of allicin and all-trans retinoic acid against CD44+ versus CD117+ melanoma cells.

BACKGROUND: All-trans retinoic acid (ATRA) is a differentiating agent that inhibits cancer cell growth during the cell cycle. However, despite its potent antitumor properties, some melanoma cells are resistant to ATRA therapy. PURPOSE: Here, we hypothesized that allicin can sensitize malignant melanoma cells to ATRA treatment. To clarify this mechanism, we determined the sensitivity to ATRA, allicin and allicin/ATRA in CD44+ and CD117+ melanoma cell subpopulations. METHODS: The CD44+and CD117+cells were sorted from A375 melanoma cell line using the magnetic-activated cell sorting (MACS). The potential anticancer effects of ATRA, allicin and allicin/ATRA were examined using cell proliferation MTT assay. In addition, flow cytometry was used to detect cell cycle arrest. The efficacy of the treatments in controlling cancer cell proliferation was assessed by quantitative realtime polymerase chain reaction (RT-PCR). RESULTS: Here, we demonstrated that CD44+ melanoma cells were more resistant to allicin and ATRA than CD117+ cells. Importantly, we observed that allicin sensitized melanoma cell to ATRA-induced cell death. The combination treatment with allicin and ATRA significantly reduced the IC50 value obtained for ATRA alone in CD44+ melanoma cells. In CD44+ cells, the IC50 value of ATRA was 37.43 ±â€¯0.54, while the IC50 value of allicin/ATRA treatment was 17.53 ±â€¯0.2 µM. Allicin treatment resulted in significant increases in the percentage of cells at the G2/M and G0/G1 phases in the CD44+ and CD117+ cells, respectively. The combination treatment caused the inhibition of CD44+ and CD117+ melanoma cells at the S phases compared to ATRA alone. Allicin, ATRA, and allicin/ATRA increased the expression of cyclin D1 mRNA in both CD44+ and CD117+ cells. Allicin combination with ATRA increased the mRNA level of RARß in CD117+ cells. Furthermore, allicin alone caused a remarkable reduction of MMP-9 mRNA expression in both CD44+ and CD117+ cells. In contrast, ATRA and the combination treatment significantly increased MMP-9 gene expression in CD44+ cells. CONCLUSION: Overall, our results indicate that allicin reinforces the ATRA-mediated inhibitory effects on CD44+ and CD117+ melanoma cells and may provide a new approach for the treatment of malignant melanoma.

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.

Uncovering Cellular retinoic acid-binding protein 2 as a potential target for rheumatoid arthritis synovial hyperplasia.

Rheumatoid arthritis (RA) is a systemic autoimmune disease including synovitis and synovial hyperplasia that contribute to joint destruction. Pivotal pathogenic mechanisms in this process are the dysregulated proliferation and apoptosis of fibroblast-like synoviocytes (FLS). Unfortunately, the mechanisms of FLS dysregulation are not completely elucidated. Here, we explored a new hypothesis based in the potent anti-proliferative and pro-apoptotic activity of retinoids in some types of cancer. Specifically, we investigated the role of retinoids and of the retinoic acid binding proteins, CRABP2 and FABP5, on the proliferation and apoptosis of FLS from RA by adding all-trans retinoic acid (ATRA) or silencing CRABP2 and FABP5. We showed an unconventional behaviour of RA FLS, which were relatively insensitive to ATRA. In effect, ATRA increased the resistance to apoptosis despite the high CRABP2/FABP5 ratio of RA FLS; and CRABP2 suppression sensitized RA FLS to Fas-induced apoptosis. This latter effect was associated with changes in expression of kinases, ASK1 up-regulation and ERK down-regulation, and increased phosphorylation of JNK. In addition, the potentiation of FLS apoptosis by CRABP2 silencing persisted in the presence of pro-inflammatory mediators, TNF e IL1ß. Therefore, the results point to CRABP2 as a potential target to decrease synovial hyperplasia in RA.

SLAB51 Probiotic Formulation Activates SIRT1 Pathway Promoting Antioxidant and Neuroprotective Effects in an AD Mouse Model.

The gut-brain axis is a bidirectional communication network functionally linking the gut and the central nervous system (CNS). Based on this, the rational manipulation of intestinal microbiota represents a novel attractive therapeutic strategy for the treatment of CNS-associated disorders. In this study, we explored the properties of a probiotic formulation (namely SLAB51) in counteracting brain oxidative damages associated with Alzheimer's disease (AD). Specifically, transgenic AD mice (3xTg-AD) were treated with SLAB51 and the effects on protein oxidation, neuronal antioxidant defence and repair systems were monitored, with the particular focus on the role of SIRT1-related pathways. We demonstrated that SLAB51 markedly reduced oxidative stress in AD mice brain by activating SIRT1-dependent mechanisms, thus representing a promising therapeutic adjuvant in AD treatment.

Molecular characterization and gene expression patterns of retinoid receptors, in normal and regenerating tissues of the sea cucumber, Holothuria glaberrima.

Retinoic acid receptors (RAR) and retinoid X receptors (RXR) are ligand-mediated transcription factors that synchronize intricate signaling networks in metazoans. Dimer formation between these two nuclear receptors mediates the recruitment of co-regulatory complexes coordinating the progression of signaling cascades during developmental and regenerative events. In the present study we identified and characterized the receptors for retinoic acid in the sea cucumber Holothuria glaberrima; a model system capable of regenerative organogenesis during adulthood. Molecular characterizations revealed the presence of three isoforms of RAR and two of RXR as a consequence of alternative splicing events. Various analyses including: primary structure sequencing, phylogenetic analysis, protein domain prediction, and multiple sequence alignment further confirmed their identity. Semiquantitative reverse transcription PCR analysis of each receptor isoform herein identified showed that the retinoid receptors are expressed in all tissues sampled: the mesenteries, respiratory trees, muscles, gonads, and the digestive tract. During regenerative organogenesis two of the receptors (RAR-L and RXR-T) showed differential expression in the posterior segment while RAR-S is differentially expressed in the anterior segment of the intestine. This work presents the first description of the components relaying the signaling for retinoic acid within this model system.