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.

Cytotoxic polyprenylated phloroglucinol derivatives from Hypericum elodeoides Choisy modulating the transactivation of RXRα.

Hyperelodione D (1), an undescribed polyprenylated phloroglucinol derivative possessing 6/6/5/5 fused tetracyclic core, together with hyperelodiones E-F (2-3), two unreported analogues bearing 6/5/5 fused tricyclic structure, were isolated from Hypericum elodeoides Choisy. Their planar structures were elucidated by spectroscopic analysis (HRESIMS, 1D and 2D NMR) and their absolute configurations were determined by comparison of experimental and calculated ECD data. The cytotoxicity and retinoid X receptor-α (RXRα) related activities of the isolates were evaluated and the plausible biogenetic pathways of 1-3 were proposed.

Structure-Activity Relationship Study Enables the Discovery of a Novel Berberine Analogue as the RXRα Activator to Inhibit Colon Cancer.

We reported recently that berberine (Ber), a traditional oriental medicine to treat gastroenteritis, binds and activates retinoid X receptor α (RXRα) for suppressing the growth of colon cancer cells. Here, we extended our studies based on the binding mode of Ber with RXRα by design, synthesis, and biological evaluation of a focused library of 15 novel Ber analogues. Among them, 3,9-dimethoxy-5,6-dihydroisoquinolino[3,2-a]isoquinolin-7-ium chloride (B-12) was identified as the optimal RXRα activator. More efficiently than Ber, B-12 bound and altered the conformation of RXRα/LBD, thereby suppressing the Wnt/ß-catenin pathway and colon cancer cell growth via RXRα mediation. In addition, B-12 not only preserved Ber's tumor selectivity but also greatly improved its bioavailability. Remarkably, in mice, B-12 did not show obvious side effects including hypertriglyceridemia as other RXRα agonists or induce hepatorenal toxicity. Together, our study describes an approach for the rational design of Ber-derived RXRα activators as novel effective antineoplastic agents for colon cancer.

Identification of the active compounds and drug targets of Chinese medicine in heart failure based on the PPARs-RXRα pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Danqi Pill (DQP), commonly known as a routinely prescribed traditional Chinese medicine (TCM), is composed of Salviae Miltiorrhizae Radix et Rhizoma and Notoginseng Radix et Rhizoma and effective in treating heart failure (HF) clinically due to their multicompound and multitarget properties. However, the exact active compounds and corresponding targets of DQP are still unknown. AIM OF THE STUDY: This study aimed to investigate active compounds and drug targets of DQP in heart failure based on the PPARs-RXRα pathway. MATERIALS AND METHODS: Network pharmacology was used to predict the compound-target interactions of DQP. Left anterior descending (LAD)-induced HF mouse model and oxygen-glucose deprivation/recovery (OGD/R)-induced H9C2 model were constructed to screen the active compounds of DQP. RESULTS: According to BATMAN-TCM (a bioinformatics analysis tool for molecular mechanism of traditional Chinese medicine we previously developed), 24 compounds in DQP were significantly enriched in the peroxisome proliferator activated receptors-retinoid X receptor α (PPARs-RXRα) pathway. Among them, Ginsenoside Rb3 (G-Rb3) had the best pharmacodynamics against OGD/R-induced loss of cell viability, and it was selected to verify the compound-target interaction. In HF mice, G-Rb3 protected cardiac functions and activated the PPARs-RXRα pathway. In vitro, G-Rb3 protected against OGD/R-induced reactive oxygen species (ROS) production, promoted the expressions of RXRα and sirtuin 3 (SIRT3), thereafter improved the intracellular adenosine triphosphate (ATP) level. Immunofluorescent staining demonstrated that G-Rb3 could activate RXRα, and facilitate RXRα shifting to the nucleus. HX531, the specific inhibitor of RXRα, could abolish the protective effects of G-Rb3 on RXRα translocation. Consistently, the effect was also confirmed on RXRα siRNA cardiomyocytes model. Moreover, surface plasmon resonance (SPR) assays identified that G-Rb3 bound directly to RXRα with the affinity of KD = 10 × 10-5 M. CONCLUSION: By integrating network pharmacology and experimental validation, we identified that as the major active compound of DQP, G-Rb3 could ameliorate ROS-induced energetic metabolism dysfunction, maintain mitochondrial function and facilitate energy metabolism via directly targeting on RXRα. This study provides a promising strategy to dissect the effective patterns for TCM and finally promote the modernization of TCM.

Hyperelodiones A-C, monoterpenoid polyprenylated acylphoroglucinols from Hypericum elodeoides, induce cancer cells apoptosis by targeting RXRα.

Hyperelodiones A-C, three undescribed monoterpenoid polyprenylated acylphloroglucinols possessing 6/6/6 fused tricyclic core, were isolated from Hypericum elodeoides Choisy. Their gross structures were elucidated by HRESIMS and NMR data. The absolute configurations of hyperelodiones A-C were assigned by their calculated and compared electronic circular dichroism (ECD) spectra combined with their common biosynthetic origin. A fluorescence quenching assay suggested that hyperelodiones A-C could bind to RXRα-LBD, whereas hyperelodione C showed the strongest interaction with a KD of 12.81 µΜ. In addition, hyperelodiones A-C dose-dependently inhibited RXRα transactivation and the growth of HeLa and MCF-7 cells. Among them, hyperelodione C showed the most potent inhibitory activities and dose-dependent PARP cleavage. Molecular docking results suggested that hyperelodione C showed a different interaction mode compared with hyperelodione A and hyperelodione B. Thus, hyperelodione C can be considered as a promising lead compound for cancer therapy, which can bind to RXRα-LBD and induce HeLa and MCF-7 cell apoptosis.

Discovery of atorvastatin as a tetramer stabilizer of nuclear receptor RXRα through structure-based virtual screening.

Retinoid X receptor alpha (RXRα), a central member of the nuclear receptor superfamily and a key regulator of many signal transduction pathways, has been an attractive drug target. We previously discovered that an N-terminally truncated form of RXRα can be induced by specific ligands to form homotetramers, which, as a result of conformational selection, forms the basis for inhibiting the nongenomic activation of RXRα. Here, we report the identification and characterization of atorvastatin as a new RXRα tetramer stabilizer by using structure-based virtual screening and demonstrate that virtual library screening can be used to aid in identifying RXRα ligands that can induce its tetramerization. In this study, docking was applied to screen the FDA-approved small molecule drugs in the DrugBank 4.0 collection. Two compounds were selected and purchased for testing. We showed that the selected atorvastatin could bind to RXRα to promote RXRα-LBD tetramerization. We also showed that atorvastatin possessed RXRα-dependent apoptotic effects. In addition, we used a chemical approach to aid in the studies of the binding mode of atorvastatin.

Glutathione deficiency alters the vitamin D-metabolizing enzymes CYP27B1 and CYP24A1 in human renal proximal tubule epithelial cells and kidney of HFD-fed mice.

Chronic kidney disease (CKD) is a worldwide public health problem with an estimated prevalence of 8.2%. This study reports glutathione deficiency, excess oxidative stress, and altered vitamin D metabolism in the kidney of mice fed a high-fat diet (HFD). The levels of GCLC and GCLM gene expression were significantly downregulated and the protein carbonylation level, a hallmark of oxidative damage, was significantly increased in the kidney of HFD-fed mice. While the levels of VD-regulatory genes 1-alpha-hydroxylase (CYP27B1), VDR, and RXRα were significantly downregulated in the kidney of mice fed a HFD, those of 24-hydroxylase (CYP24A1) were significantly elevated. In vitro, GSH deficiency per se causes excess oxidative damage (protein carbonylation), and significantly decreases the levels of VD-regulatory genes (CYP27B1, VDR, and RXRα), but increases levels of CYP24A1 in human renal proximal tubule epithelial cells (RPTEC), similar to findings in the kidney of HFD-fed diabetic mice. L-cysteine supplementation restores GSH and prevents oxidative damage in RPTEC. These studies suggest a potential role of GSH precursor in reducing excess oxidative stress and renal injury that commonly accompanies obesity/diabetes.

Gestational Vitamin D Supplementation Leads to Reduced Perinatal RXRA DNA Methylation: Results From the MAVIDOS Trial.

We have previously demonstrated inverse associations between maternal 25(OH)-vitamin D status and perinatal DNA methylation at the retinoid-X-receptor-alpha (RXRA) locus and between RXRA methylation and offspring bone mass. In this study, we used an existing randomized trial to test the hypothesis that maternal gestational vitamin D supplementation would lead to reduced perinatal RXRA locus DNA methylation. The Maternal Vitamin D Osteoporosis Study (MAVIDOS) was a multicenter, double-blind, randomized, placebo-controlled trial of 1000 IU/day cholecalciferol or matched placebo from 14 weeks' gestation until delivery. Umbilical cord (fetal) tissue was collected at birth and frozen at -80°C (n = 453). Pyrosequencing was used to undertake DNA methylation analysis at 10 CpG sites within the RXRA locus (identified previously). T tests were used to assess differences between treatment groups in methylation at the three most representative CpG sites. Overall, methylation levels were significantly lower in the umbilical cord from offspring of cholecalciferol-supplemented mothers, reaching statistical significance at four CpG sites, represented by CpG5: mean difference in % methylation between the supplemented and placebo groups was -1.98% (95% CI, -3.65 to -0.32, p = 0.02). ENCODE (Encyclopedia of DNA Elements) evidence supports the functionality of this locus with strong DNase hypersensitivity and enhancer chromatin within biologically relevant cell types including osteoblasts. Enrichment of the enhancer-related H3K4me1 histone mark is also seen in this region, as are binding sites for a range of transcription factors with roles in cell proliferation, response to stress, and growth factors. Our findings are consistent with previous observational results and provide new evidence that maternal gestational supplementation with cholecalciferol leads to altered perinatal epigenetic marking, informing mechanistic understanding of early life mechanisms related to maternal vitamin D status, epigenetic marks, and bone development. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Epigallocatechin-3-gallate inhibited cancer stem cell-like properties by targeting hsa-mir-485-5p/RXRα in lung cancer.

Non-small-cell lung cancer (NSCLC) appears to be a significant threat to public health worldwide. MicroRNAs have been identified as significant regulators for the development of NSCLC. Previous reports have suggested that hsa-mir-485-5p is dysregulated in various cancers. RXRα, as a kind of nuclear receptor, is an effective target of cancer treatment. Cancer stem cells (CSCs) are recognized as the main cause for tumor metastasis, recurrence, and chemotherapy resistance. However, the mechanism by which hsa-mir-485-5p and RXRα modulate CSCs in NSCLC remains unknown. Here, we found that hsa-mir-485-5p was decreased in serum samples from patients with NSCLC and NSCLC cells. Meanwhile, epigallocatechin-3-gallate (EGCG), an effective anticancer compound extracted from green tea, can enhance hsa-mir-485-5p expression. Hsa-mir-485-5p mimics markedly inhibited NSCLC cell growth and induced cell apoptosis. However, inhibition of hsa-mir-485-5p significantly enriched CSC-like traits. Moreover, bioinformatics analysis predicted the binding correlation between hsa-mir-485-5p and RXRα, which was confirmed by a dual-luciferase reporter assay. We observed that RXRα was increased in NSCLC and EGCG could inhibit RXRα levels dose dependently. In addition, RXRα upregulation or activation expanded the CSC-like properties of NSCLC cells, whereas RXRα inhibition or inactivation could exert a reverse phenomenon. Consistently, in vivo experiments also validated that EGCG could repress the CSC-like characteristics by modulating the hsa-mir-485-5p/RXRα axis. Our findings may reveal a novel molecular mechanism for the treatment of NSCLC.

Berberine binds RXRα to suppress ß-catenin signaling in colon cancer cells.

Berberine, an isoquinoline alkaloid, is a traditional oriental medicine used to treat diarrhea and gastroenteritis. Recently, we reported that it could inhibit the growth of intestinal polyp in animals and in patients with the familial adenomatous polyposis by downregulating ß-catenin signaling. However, the intracellular target mediating the effects of berberine remains elusive. Here, we provide evidence that berberine inhibits ß-catenin function via directly binding to a unique region comprising residues Gln275, Arg316 and Arg371 in nuclear receptor retinoid X receptor alpha (RXRα), where berberine concomitantly binding to and synergistically activating RXRα with 9-cis-retinoic acid (9-cis-RA), a natural ligand binding to the classical ligand-binding pocket of RXRα. Berberine binding promotes RXRα interaction with nuclear ß-catenin, leading to c-Cbl mediated degradation of ß-catenin, and consequently inhibits the proliferation of colon cancer cells. Furthermore, berberine suppresses the growth of human colon carcinoma xenograft in nude mice in an RXRα-dependent manner. Together, our study not only identifies RXRα as a direct protein target for berberine but also dissects their binding mode and validates that berberine indeed suppresses ß-catenin signaling and cell growth in colon cancer via binding RXRα, which provide new strategies for the design of new RXRα-based antitumor agents and drug combinations.

Development of a Magnetic Microbead Affinity Selection Screen (MagMASS) Using Mass Spectrometry for Ligands to the Retinoid X Receptor-α.

To overcome limiting factors in mass spectrometry-based screening methods such as automation while still facilitating the screening of complex mixtures such as botanical extracts, magnetic microbead affinity selection screening (MagMASS) was developed. The screening process involves immobilization of a target protein on a magnetic microbead using a variety of possible chemistries, incubation with mixtures of molecules containing possible ligands, a washing step that removes non-bound compounds while a magnetic field retains the beads in the microtiter well, and an organic solvent release step followed by LC-MS analysis. Using retinoid X receptor-α (RXRα) as an example, which is a nuclear receptor and target for anti-inflammation therapy as well as cancer treatment and prevention, a MagMASS assay was developed and compared with an existing screening assay, pulsed ultrafiltration (PUF)-MS. Optimization of MagMASS involved evaluation of multiple protein constructs and several magnetic bead immobilization chemistries. The full-length RXRα construct immobilized with amylose beads provided optimum results. Additional enhancements of MagMASS were the application of 96-well plates to enable automation, use of UHPLC instead of HPLC for faster MS analyses, and application of metabolomics software for faster, automated data analysis. Performance of MagMASS was demonstrated using mixtures of synthetic compounds and known ligands spiked into botanical extracts. Graphical Abstract ᅟ.

The effect of Chinese herbs and its effective components on coronary heart disease through PPARs-PGC1α pathway.

BACKGROUND: DanQi pill (DQP) is prescribed widely in China and has definite cardioprotective effect on coronary heart disease. Our previous studies proved that DQP could effectively regulate plasma levels of high density lipoprotein (HDL) and low density lipoprotein (LDL). However, the regulatory mechanisms of DQP and its major components Salvianolic acids and Panax notoginseng saponins (DS) on lipid metabolism disorders haven't been comprehensively studied so far. METHODS: Rat model of coronary heart disease was induced by left anterior descending (LAD) artery ligation operations. Rats were divided into sham, model, DQP treated, DS treated and positive drug (clofibrate) treated groups. At 28 days after surgery, cardiac functions were assessed by echocardiography. Expressions of transcription factors and key molecules in energy metabolism pathway were measured by reverse transcriptase polymerase chain reaction or western blotting. RESULTS: In ischemic heart model, cardiac functions were severely injured but improved by treatments of DQP and DS. Expression of LPL was down-regulated in model group. Both DQP and DS could up-regulate the mRNA expression of LPL. Membrane proteins involved in lipid transport and uptake, such as FABP4 and CPT-1A, were down-regulated in ischemic heart tissues. Treatment with DQP and DS regulated lipid metabolisms by up-regulating expressions of FABP4 and CPT-1A. DQP and DS also suppressed expression of cytochrome P450. Furthermore, transcriptional factors, such as PPARα, PPARγ, RXRA and PGC-1α, were down-regulated in ischemic model group. DQP and DS could up-regulate expressions of these factors. However, DS showed a better efficacy than DQP on PGC-1α, a coactivator of PPARs. Key molecules in signaling pathways such as AKT1/2, ERK and PI3K were also regulated by DQP and DS simultaneously. CONCLUSIONS: Salvianolic acids and Panax notoginseng are the major effective components of DanQi pill in improving lipid metabolism in ischemic heart model. The effects may be mediated by regulating transcriptional factors such as PPARs, RXRA and PGC-1α.

Upregulation of rat liver PPARα-FGF21 signaling by a docosahexaenoic acid and thyroid hormone combined protocol.

Prevention of ischemia-reperfusion liver injury is achieved by a combined omega-3 and thyroid hormone (T3 ) protocol, which may involve peroxisome-proliferator activated receptor-α (PPAR-α)-fibroblast growth factor 21 (FGF21) signaling supporting energy requirements. Combined docosahexaenoic acid (DHA; daily doses of 300 mg/kg for 3 days) plus 0.05 mg T3 /kg given to fed rats elicited higher hepatic DHA contents and serum T3 levels, increased PPAR-α mRNA and its DNA binding, with higher mRNA expression of the PPAR-α target genes for carnitine-palmitoyl transferase 1α, acyl-CoA oxidase, and 3-hydroxyl-3-methylglutaryl-CoA synthase 2, effects that were mimicked by 0.1 mg T3 /kg given alone or by the PPAR-α agonist WY-14632. Under these conditions, the mRNA expression of retinoic X receptor-α (RXR-α) is also increased, with concomitant elevation of the hepatic mRNA and protein FGF21 levels and those of serum FGF21. It is concluded that PPAR-α-FGF21 induction by DHA combined with T3 may involve ligand activation of PPAR-α by DHA and enhanced expression of PPAR-α by T3 , with consequent upregulation of the FGF21 that is controlled by PPAR-α. Considering the beneficial effects of PPAR-α-FGF21 signaling on carbohydrate and lipid metabolism, further investigations are required to clarify its potential therapeutic applications in human metabolic disorders. © 2016 BioFactors, 42(6):638-646, 2016.

Influence of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 on expression of P-glycoprotein and cytochrome P450 3A in sheep.

In order to improve calcium and phosphorus balance, beef cattle and dairy cows can be supplemented with vitamin D. However, different vitamin D metabolites have been shown to increase expression of P-glycoprotein (P-gp, MDR1, ABCB1) and cytochrome P450 3A (CYP3A) in rodents as well as in cell culture systems. As such interferences might have an impact on pharmacokinetics of some drugs widely-used in veterinary medicine, we investigated the expression of P-gp, CYP3A, vitamin D receptor (VDR), pregnane X receptor (PXR) and retinoid X receptor α (RXRα) in sheep either treated orally with 6µg/kg body weight (BW) 25-hydroxyvitamin D3 (OHD3) for ten days before sacrifice or 12h after intravenous injection of 0.5µg/kg BW 1,25-dihydroxyvitamin D3 (1,25- (OH)2D3). Down-regulation of ruminal, jejunal and hepatic, but not renal P-gp could be found with 25-OHD3 supplementation. Interestingly, this effect on P-gp was not observed in tissues from 1,25-(OH)2D3-treated sheep. In contrast, 1,25-(OH)2D3 induced a significant up-regulation of renal and jejunal CYP3A expression, while 25-OHD3 had no impact. Renal expression of VDR and PXR was also increased by treatment with 1,25-(OH)2D3, while jejunal PXR expression was only stimulated in sheep supplemented with 25-OHD3. Either treatments increased renal, but not ruminal, jejunal or hepatic expression of RXRα. These results demonstrate that the impact of large doses of vitamin D metabolites on different target organs and potential interactions with other medications should be further investigated in vitro and in vivo to understand the effects of vitamin D metabolites on metabolism and excretion pathways in livestock.

Prospective associations between vitamin D status, vitamin D-related gene polymorphisms, and risk of tobacco-related cancers.

BACKGROUND: Experimental evidence has suggested that vitamin D may be protective against tobacco-related cancers through the inhibition of the formation of tumors induced by tobacco carcinogens. To our knowledge, only one previous epidemiologic study investigated the association between vitamin D status and tobacco-related cancer risk, and no study has focused on vitamin D-related gene polymorphisms. OBJECTIVE: Our objective was to prospectively study the association between plasma 25-hydroxyvitamin D [25(OH)D] concentrations, vitamin D-related gene polymorphisms, and risk of tobacco-related cancers. DESIGN: A total of 209 tobacco-related cancers were diagnosed within the SU.VI.MAX (Supplémentation en vitamines et minéraux antioxydants) cohort (1994-2007) and were matched with 418 controls as part of a nested case-control study. Tobacco-related cancers (i.e., cancers for which tobacco is one of the risk factors) included several sites in the respiratory, digestive, reproductive, and urinary systems. Total plasma 25(OH)D was assessed with the use of an electrochemoluminescent assay. Polymorphisms were determined with the use of a TaqMan assay. Conditional logistic regression models were computed. RESULTS: A 25(OH)D concentration ≥30 ng/mL was associated with reduced risk of tobacco-related cancers (OR for ≥30 compared with <30 ng/mL: 0.59; 95% CI 0.35, 0.99; P = 0.046). This association was observed in former and current smokers (OR for ≥30 compared with <30 ng/mL: 0.43; 95% CI: 0.23, 0.84; P = 0.01) but not in never smokers (P = 0.8). The vitamin D receptor (VDR) FokI AA genotype and retinoid X receptor (RXR) rs7861779 TT genotype were associated with increased risk of tobacco-related cancers [OR for homozygous mutant type (MT) compared with wild type (WT): 1.87; 95% CI: 1.08, 3.23; P-trend = 0.02; OR for heterozygous type (HT) plus MT compared with WT: 1.60; 95% CI: 1.07, 2.38; P = 0.02]. CONCLUSIONS: In this prospective study, high vitamin D status [25(OH)D concentration ≥30 ng/mL] was associated with decreased risk of tobacco-related cancers, especially in smokers. These results, which are supported by mechanistic plausibility, suggest that vitamin D may contribute to the prevention of tobacco-induced cancers in smokers and deserve additional investigation. The SU.VI.MAX trial was registered at clinicaltrials.gov as NCT00272428.

Trimodal Therapy: Combining Hyperthermia with Repurposed Bexarotene and Ultrasound for Treating Liver Cancer.

Repurposing of existing cancer drugs to overcome their physical limitations, such as insolubility, represents an attractive strategy to achieve enhanced therapeutic efficacy and broaden the range of clinical applications. Such an approach also promises to offer substantial cost savings in drug development efforts. Here we repurposed FDA-approved topical agent bexarotene (Targretin), currently in limited use for cutaneous manifestations of T-cell lymphomas, and re-engineer it for use in solid tumor applications by forming self-assembling nanobubbles. Physico-chemical characterization studies of the novel prodrug nanobubbles demonstrated their stability, enhanced target cell internalization capability, and highly controlled release profile in response to application of focused ultrasound energy. Using an in vitro model of hepatocellular carcinoma and an in vivo large animal model of liver ablation, we demonstrate the effectiveness of bexarotene prodrug nanobubbles when used in conjunction with catheter-based ultrasound, thereby highlighting the therapeutic promise of this trimodal approach.

Neuroendocrine factors regulate retinoic acid receptors in normal and hypoplastic lung development.

KEY POINTS: Retinoic acid (RA) and ghrelin levels are altered in human hypoplastic lungs when compared to healthy lungs. Although considerable data have been obtained about RA, ghrelin and bombesin in the congenital diaphragmatic hernia (CDH) rat model, neuroendocrine factors have never been associated with the RA signalling pathway in this animal model. In this study, the interaction between neuroendocrine factors and RA was explored in the CDH rat model. The authors found that normal fetal lung explants treated with RA, bombesin and ghrelin showed an increase in lung growth. Hypoplastic lungs presented higher expression levels of the RA receptors α and γ. Moreover bombesin and ghrelin supplementation, in vitro, to normal lungs increased RA receptor α/γ expression whereas administration of bombesin and ghrelin antagonists to normal and hypoplastic lungs decreased it. These data reveal for the first time that there is a link between neuroendocrine factors and RA, and that neuroendocrine factors sensitise the lung to the RA action through RA receptor modulation. ABSTRACT: Congenital diaphragmatic hernia (CDH) is characterised by a spectrum of lung hypoplasia and consequent pulmonary hypertension, leading to high morbidity and mortality rates. Moreover, CDH has been associated with an increase in the levels of pulmonary neuroendocrine factors, such as bombesin and ghrelin, and a decrease in the action of retinoic acid (RA). The present study aimed to elucidate the interaction between neuroendocrine factors and RA. In vitro analyses were performed on Sprague-Dawley rat embryos. Normal lung explants were treated with bombesin, ghrelin, a bombesin antagonist, a ghrelin antagonist, dimethylsulfoxide (DMSO), RA dissolved in DMSO, bombesin plus RA and ghrelin plus RA. Hypoplastic lung explants (nitrofen model) were cultured with bombesin, ghrelin, bombesin antagonist or ghrelin antagonist. The lung explants were analysed morphometrically, and retinoic acid receptor (RAR) α, ß and γ expression levels were assessed via Western blotting. Immunohistochemistry analysis of RAR was performed in normal and hypoplastic lungs 17.5 days post-conception (dpc). Compared with the controls, hypoplastic lungs exhibited significantly higher RARα/γ expression levels. Furthermore considering hypoplastic lungs, bombesin and ghrelin antagonists decreased RARα/γ expression. Normal lung explants (13.5 dpc) treated with RA, bombesin plus RA, ghrelin plus RA, bombesin or ghrelin exhibited increased lung growth. Moreover, bombesin and ghrelin increased RARα/γ expression levels, whereas the bombesin and ghrelin antagonists decreased RARα/γ expression. This study demonstrates for the first time that neuroendocrine factors function as lung growth regulators, sensitising the lung to the action of RA through up-regulation of RARα and RARγ.

Molecular characterization, transcriptional activity and nutritional regulation of peroxisome proliferator activated receptor gamma in Nile tilapia (Oreochromis niloticus).

Peroxisome proliferator activated receptor gamma (PPARγ) is a master regulator in lipid metabolism and widely exists in vertebrates. However, the molecular structure and transcriptional activity of PPARγ in fish are still unclear. This study cloned PPARγ from Nile tilapia (Oreochromis niloticus) referred as NtPPARγ and transfected the NtPPARγ plasmids into HEK-293 cells to explore its mechanism of transcriptional regulation in fish. The expression of NtPPARγ was compared in fed and fasted fish. Two transcripts of NtPPARγ varied at the 5'-untranslated region and the DNA binding domain was highly conserved. Thirty-nine amino acid residues in the ligand binding domain in Nile tilapia were different from those in human. Two transcripts showed different expression profiles in 11 tissues, but both were highly expressed in liver, intestine and kidney. The transcriptional activity assay showed that NtPPARγ collaborates with retinoid X-receptor α (NtRXRα) to regulate the expression of Nile tilapia fatty acid binding protein 4 (FABP4), the compartment of which have been identified as the target gene of PPARγ in human. In the fish fasting trial, the mRNA expression of NtPPARγ1 and NtPPARγ2 in intestine and liver at 3h post-feeding (HPF) was lower than those at 8 HPF, 24 HPF and in fish fasted for 36h, but was relatively stable in kidney among different feeding treatments. In conclusion, the DNA binding domain in PPARγ was highly conserved, while the ligand binding domain was moderately conserved. In Nile tilapia, the PPARγ collaborates with RXRα to perform transcriptional regulation of FABP4 at least in vitro. The plasmid system established in this study along with a cell line from Nile tilapia will be useful tools for the further functional study of PPARγ in fish.

Identification of a naturally occurring retinoid X receptor agonist from Brazilian green propolis.

BACKGROUND: Brazilian green propolis (BGP), a resinous substance produced from Baccharis dracunculifolia by Africanized honey bees (Apis mellifera), is used as a folk medicine. Our present study explores the retinoid X receptor (RXR) agonistic activity of BGP and the identification of an RXR agonist in its extract. METHODS: RXRα agonistic activity was evaluated using a luciferase reporter gene assay. Isolation of the RXRα agonist from the ethanolic extract of BGP was performed using successive silica gel and a reversed phase column chromatography. The interaction between the isolated RXRα agonist and RXRα protein was predicted by a receptor-ligand docking simulation. The nuclear receptor (NR) cofactor assay was used to estimate whether the isolated RXRα agonist bound to various NRs, including RXRs and peroxisome proliferator-activated receptors (PPARs). We further examined its effect on adipogenesis in 3T3-L1 fibroblasts. RESULTS: We identified drupanin as an RXRα agonist with an EC50 value of 4.8 ± 1.0 µM. Drupanin activated three RXR subtypes by a similar amount and activated PPARγ moderately. Additionally, drupanin induced adipogenesis and elevated aP2 mRNA levels in 3T3-L1 fibroblasts. CONCLUSIONS: Drupanin, a component of BGP, is a novel RXR agonist with slight PPARγ agonistic activity. GENERAL SIGNIFICANCE: This study revealed for the first time that BGP activates RXR and drupanin is an RXR agonist in its extract.

Identification of spliced mRNA isoforms of retinoid X receptor (RXR) in the Oriental freshwater prawn Macrobrachium nipponense.

Retinoid X receptors (RXR) are members of the nuclear receptor family that are conserved from invertebrates to vertebrates, and they play an essential role in regulating reproductive maturation, molting, and embryo development. In this study, five RXR isoforms, named RXRL2 (L, long form), RXRL3, RXRS1 (S, short form), RXRS2, and RXRS3, containing six domains from A to F, were cloned from the prawn Macrobrachium nipponense using 5'- and 3'- rapid amplification of cDNA ends. Differences among their structures were observed not only in the D and E domains but also in the A/B domain, which were previously found in insects but not in crustaceans. This is the first report to show that differences occur in the A/B domain of RXR in crustaceans. RXR expressions were also examined in various tissues including the ovary, testis, muscle, hepatopancreas, heart, gill, stomach, intestine, and cuticle. Expression pattern investigations indicated that the five isoforms were differentially expressed. RXRS3 was only detected in the ovary, and the other RXRs were abundant in the ovary and testis. These data suggested that RXR mediates a series of processes related to reproduction.