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1.
Gene ; 763: 145058, 2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-32798635

RESUMO

BACKGROUND: The nuclear receptors Rev-erb alpha and Rev-erb beta are transcription factors that regulate the function of genes in glucose and lipid metabolism, and they also form a link between circadian rhythm and metabolism. We evaluated the variations in Rev-erb alpha and Rev-erb beta genes together with biochemical parameters as risk factors in type 2 diabetic (T2DM) patients. METHODS: Molecular analyses of Rev-erb alpha and Rev-erb beta genes were performed on genomic DNA by using next-generation sequencing in 42 T2DM patients (21 obese and 21 non-obese) and 66 healthy controls. RESULTS: We found 26 rare mutations in the study groups, including 13 missense mutations, 9 silent mutations, 3 5'UTR variations, and a 3'UTR variation, of which 9 were novel variations (5 missense and 3 silent and 1 5'UTR). Six common variations were also found in the Rev-erb genes; Rev-erb beta Chr3:24003765 A > G, Rev-erb beta rs924403442 (Chr3:24006717) G > T, Rev-erb alpha Chr17:38253751 T > C, Rev-erb alpha rs72836608 C > A, Rev-erb alpha rs2314339 C > T and Rev-erb alpha rs2102928 C > T. Of these, Rev-erb beta Chr3:24003765 A > G was a novel missense mutation (p.Q197R), while others were identified as intronic variants. T2DM patients with Rev-erb beta rs924403442 T allele had lower body surface area (BSA) than noncarriers (GG genotype) (p = 0.039). Rev-erb alpha rs72836608 A allele and Rev-erb alpha rs2314339 CC genotype were associated with decreased serum HDL-cholesterol levels in T2DM patients (p = 0.025 and p = 0.027, respectively). In our study, different effects of Rev-erbs polymorphisms were found according to gender and presence of obesity. Rev-erb alpha rs72836608 (C > A) and rs2314339 (C > T) and Rev-erb alpha rs2102928 (C > T) were associated with low HDL-C levels in male T2DM patients. In female patients, Rev-erb alpha rs2102928 (C > T) was associated with high microalbuminuria and Rev-erb beta rs9244403442 G > T was associated with low HDL and high BSA values. In addition, Rev-erb alpha Chr17: 38,253,751 (T > C), rs72836608 (C > A), and rs2314339 (C > T) and Rev-erb beta Chr3:24003765 (A > G) were associated with increased serum GGT levels in obese T2DM patients. In non-obese patients, Rev-erbs SNPs had no effect on serum GGT levels. CONCLUSION: Our findings indicate that variations in the Rev-erb alpha and Rev-erb beta genes can affect metabolic changes in T2DM and these effects may vary depending on gender and obesity.


Assuntos
Diabetes Mellitus Tipo 2/genética , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Polimorfismo de Nucleotídeo Único , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Repressoras/genética , Diabetes Mellitus Tipo 2/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
2.
J Oleo Sci ; 69(9): 1077-1085, 2020 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-32788520

RESUMO

There is growing research interest in the hypocholesterolemic effect of various food components such as polyphenols. In this study, we examined the effects of oligonol-a low-molecular weight polyphenol extracted from lychee fruit-on cholesterol metabolism in rats under short-term administration. Administration of oligonol for 3 days significantly increased cecum weight and decreased cecal n-butyric acid concentrations in rats. Oligonol also significantly lowered the levels of hepatic cholesterol and increased the levels of total neutral steroids excreted in the feces. It also increased fecal ß-muricholic acid significantly, whereas the levels of total acidic steroids remained unchanged. Gene expression of hepatic CYP7A1 (cytochrome P450 family 7 subfamily A member 1) significantly increased following the administration of oligonol. This increase could be ascribed to changes in the expression of farnesoid X receptor, small heterodimer partner, and fibroblast growth factor 15 in ileum. Our data suggest that oligonol induces hypocholesterolemic effects through the inhibition of biliary cholesterol absorption from the intestine and the upregulation of cholesterol catabolism in rats even following short-term administration. Therefore, oligonol may be an important food component for reducing cholesterol level.


Assuntos
Catequina/análogos & derivados , Colesterol/metabolismo , Litchi/química , Fenóis/isolamento & purificação , Fenóis/farmacologia , Animais , Butiratos/metabolismo , Catequina/administração & dosagem , Catequina/isolamento & purificação , Catequina/farmacologia , Colesterol 7-alfa-Hidroxilase/genética , Colesterol 7-alfa-Hidroxilase/metabolismo , Expressão Gênica/efeitos dos fármacos , Íleo/metabolismo , Fígado/metabolismo , Masculino , Peso Molecular , Fenóis/administração & dosagem , Polifenóis , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Tempo
3.
Nat Commun ; 11(1): 3306, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620754

RESUMO

The endoplasmic reticulum (ER) is selectively degraded by autophagy (ER-phagy) through proteins called ER-phagy receptors. In Saccharomyces cerevisiae, Atg40 acts as an ER-phagy receptor to sequester ER fragments into autophagosomes by binding Atg8 on forming autophagosomal membranes. During ER-phagy, parts of the ER are morphologically rearranged, fragmented, and loaded into autophagosomes, but the mechanism remains poorly understood. Here we find that Atg40 molecules assemble in the ER membrane concurrently with autophagosome formation via multivalent interaction with Atg8. Atg8-mediated super-assembly of Atg40 generates highly-curved ER regions, depending on its reticulon-like domain, and supports packing of these regions into autophagosomes. Moreover, tight binding of Atg40 to Atg8 is achieved by a short helix C-terminal to the Atg8-family interacting motif, and this feature is also observed for mammalian ER-phagy receptors. Thus, this study significantly advances our understanding of the mechanisms of ER-phagy and also provides insights into organelle fragmentation in selective autophagy of other organelles.


Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Família da Proteína 8 Relacionada à Autofagia/química , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/genética , Sítios de Ligação/genética , Estresse do Retículo Endoplasmático/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Mutação , Ligação Proteica , Domínios Proteicos , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
4.
PLoS One ; 15(7): e0236520, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32730286

RESUMO

In eukaryotic cells, phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of the lipid bilayer. Budding yeast contains five flippases, of which Cdc50p-Drs2p and Neo1p are primarily involved in membrane trafficking in endosomes and Golgi membranes. The ANY1/CFS1 gene was identified as a suppressor of growth defects in the neo1Δ and cdc50Δ mutants. Cfs1p is a membrane protein of the PQ-loop family and is localized to endosomal/Golgi membranes, but its relationship to phospholipid asymmetry remains unknown. The neo1Δ cfs1Δ mutant appears to function normally in membrane trafficking but may function abnormally in the regulation of phospholipid asymmetry. To identify a gene that is functionally relevant to NEO1 and CFS1, we isolated a mutation that is synthetically lethal with neo1Δ cfs1Δ and identified ERD1. Erd1p is a Golgi membrane protein that is involved in the transport of phosphate (Pi) from the Golgi lumen to the cytoplasm. The Neo1p-depleted cfs1Δ erd1Δ mutant accumulated plasma membrane proteins in the Golgi, perhaps due to a lack of phosphatidylinositol 4-phosphate. The Neo1p-depleted cfs1Δ erd1Δ mutant also exhibited abnormal structure of the endoplasmic reticulum (ER) and induced an unfolded protein response, likely due to defects in the retrieval pathway from the cis-Golgi region to the ER. Genetic analyses suggest that accumulation of Pi in the Golgi lumen is responsible for defects in Golgi functions in the Neo1p-depleted cfs1Δ erd1Δ mutant. Thus, the luminal ionic environment is functionally relevant to phospholipid asymmetry. Our results suggest that flippase-mediated phospholipid redistribution and luminal Pi concentration coordinately regulate Golgi membrane functions.


Assuntos
Complexo de Golgi/metabolismo , Fosfatos/metabolismo , Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/genética , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Mutação , Proteínas de Transferência de Fosfolipídeos/genética , Receptores Citoplasmáticos e Nucleares/genética , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Resposta a Proteínas não Dobradas
5.
Gene ; 751: 144779, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32428697

RESUMO

In insects, nuclear receptors (NRs) including EcR (NR1H1), USP (NR2B4), E75 (NR1D3), HR3 (NR1F), HR4 (NR6) and FTZ-F1 (NR5A3) mediate the 20-hydroxyecdysone (20E) signaling cascade to play a critical role during larval metamorphosis. In this present paper, we focused on hormone receptor 38 (HR38) in Leptinotarsa decemlineata, the only insect homolog of the NR4A subclass. RNA interference (RNAi) of LdHR38 in the penultimate (third) instar larvae reduced the expression of an ecdysteroidogenesis gene and declined the titer of 20E. Knockdown of LdHR38 intensified the expression of LdUSP, LdE75, LdE74, LdE93, LdBroad and LdHR3, whereas repressed the transcription of LdFTZ-F1. Disruption of 20E signaling inhibited chitin biosynthesis in the larval cuticle. Approximately 25% of the LdHR38 RNAi larvae died, around 40% of the resultant larvae remained as prepupae or become deformed pupae. The body surface of the HR38 depleted abnormal prepupae and pupae looked wet, just like the cuticle being covered with a layer of liquid. Moreover, the increase of larval mortality, and the impairment of pupation and emergence exhibited dose-dependent manners. Furthermore, silencing LdHR38 at the final (fourth) instar caused similar but less severe impairment of pupation. Dietary supplement with 20E for the third instar larvae did not rescue the high larval death and only slightly alleviated the low pupation rate in the LdHR38 RNAi hypomorphs. Accordingly, we propose that HR38 is necessary for tune of ecdysteroidogenesis and for mediation of 20E signaling during metamorphosis in L. decemlineata.


Assuntos
Besouros/crescimento & desenvolvimento , Proteínas de Insetos/fisiologia , Metamorfose Biológica , Receptores Citoplasmáticos e Nucleares/fisiologia , Animais , Quitina/biossíntese , Besouros/genética , Besouros/metabolismo , Ecdisterona/fisiologia , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/genética , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Pupa/genética , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , Interferência de RNA , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Transdução de Sinais
6.
Nat Commun ; 11(1): 1720, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32249768

RESUMO

Nuclear localization of PTEN is essential for its tumor suppressive role, and loss of nuclear PTEN is more prominent than cytoplasmic PTEN in many kinds of cancers. However, nuclear PTEN-specific regulatory mechanisms were rarely reported. Based on the finding that nuclear PTEN is more unstable than cytoplasmic PTEN, here we identify that F-box only protein 22 (FBXO22) induces ubiquitylation of nuclear but not cytoplasmic PTEN at lysine 221, which is responsible for the degradation of nuclear PTEN. FBXO22 plays a tumor-promoting role by ubiquitylating and degrading nuclear PTEN. In accordance, FBXO22 is overexpressed in various cancer types, and contributes to nuclear PTEN downregulation in colorectal cancer tissues. Cumulatively, our study reports the mechanism to specifically regulate the stability of nuclear PTEN, which would provide the opportunity for developing therapeutic strategies aiming to achieve complete reactivation of PTEN as a tumor suppressor.


Assuntos
Carcinogênese/genética , Núcleo Celular/metabolismo , Transformação Celular Neoplásica/genética , Neoplasias Colorretais/metabolismo , Proteínas F-Box/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Linhagem Celular Tumoral , Cromatografia Líquida , Neoplasias Colorretais/genética , Citoplasma/metabolismo , Proteínas F-Box/genética , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos SCID , PTEN Fosfo-Hidrolase/química , PTEN Fosfo-Hidrolase/genética , RNA Interferente Pequeno , Receptores Citoplasmáticos e Nucleares/genética , Transdução de Sinais/genética , Espectrometria de Massas em Tandem , Análise Serial de Tecidos , Transplante Heterólogo , Ubiquitinação
7.
PLoS Genet ; 16(4): e1008687, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282814

RESUMO

Environment shapes development through a phenomenon called developmental plasticity. Deciphering its genetic basis has potential to shed light on the origin of novel traits and adaptation to environmental change. However, molecular studies are scarce, and little is known about molecular mechanisms associated with plasticity. We investigated the gene regulatory network controlling predatory vs. non-predatory dimorphism in the nematode Pristionchus pacificus and found that it consists of genes of extremely different age classes. We isolated mutants in the conserved nuclear hormone receptor nhr-1 with previously unseen phenotypic effects. They disrupt mouth-form determination and result in animals combining features of both wild-type morphs. In contrast, mutants in another conserved nuclear hormone receptor nhr-40 display altered morph ratios, but no intermediate morphology. Despite divergent modes of control, NHR-1 and NHR-40 share transcriptional targets, which encode extracellular proteins that have no orthologs in Caenorhabditis elegans and result from lineage-specific expansions. An array of transcriptional reporters revealed co-expression of all tested targets in the same pharyngeal gland cell. Major morphological changes in this gland cell accompanied the evolution of teeth and predation, linking rapid gene turnover with morphological innovations. Thus, the origin of feeding plasticity involved novelty at the level of genes, cells and behavior.


Assuntos
Evolução Molecular , Proteínas de Helminto/genética , Comportamento Predatório , Receptores Citoplasmáticos e Nucleares/genética , Rabditídios/genética , Animais , Sequência Conservada , Redes Reguladoras de Genes , Proteínas de Helminto/metabolismo , Boca/anatomia & histologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Rabditídios/anatomia & histologia , Rabditídios/fisiologia , Análise de Célula Única
8.
PLoS One ; 15(2): e0224644, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32101552

RESUMO

Polybrominated diphenyl ethers (PBDEs) were formally used as flame-retardants and are chemically stable, lipophlic persistent organic pollutants which are known to bioaccumulate in humans. Although its toxicities are well characterized, little is known about the changes in transcriptional regulation caused by PBDE exposure. Long non-coding RNAs (lncRNAs) are increasingly recognized as key regulators of transcriptional and translational processes. It is hypothesized that lncRNAs can regulate nearby protein-coding genes (PCGs) and changes in the transcription of lncRNAs may act in cis to perturb gene expression of its neighboring PCGs. The goals of this study were to 1) characterize PCGs and lncRNAs that are differentially regulated from exposure to PBDEs; 2) identify PCG-lncRNA pairs through genome annotation and predictive binding tools; and 3) determine enriched canonical pathways caused by differentially expressed lncRNA-PCGs pairs. HepaRG cells, which are human-derived hepatic cells that accurately represent gene expression profiles of human liver tissue, were exposed to BDE-47 and BDE-99 at a dose of 25 µM for 24 hours. Differentially expressed lncRNA-PCG pairs were identified through DESeq2 and HOMER; significant canonical pathways were determined through Ingenuity Pathway Analysis (IPA). LncTar was used to predict the binding of 19 lncRNA-PCG pairs with known roles in drug-processing pathways. Genome annotation revealed that the majority of the differentially expressed lncRNAs map to PCG introns. PBDEs regulated overlapping pathways with PXR and CAR such as protein ubiqutination pathway and peroxisome proliferator-activated receptor alpha-retinoid X receptor alpha (PPARα-RXRα) activation but also regulate distinctive pathways involved in intermediary metabolism. PBDEs uniquely down-regulated GDP-L-fucose biosynthesis, suggesting its role in modifying important pathways involved in intermediary metabolism such as carbohydrate and lipid metabolism. In conclusion, we provide strong evidence that PBDEs regulate both PCGs and lncRNAs in a PXR/CAR ligand-dependent and independent manner.


Assuntos
Retardadores de Chama/farmacologia , Perfilação da Expressão Gênica/métodos , Éteres Difenil Halogenados/farmacologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , RNA Longo não Codificante/metabolismo , Metabolismo dos Carboidratos , Linhagem Celular , Retardadores de Chama/administração & dosagem , Regulação da Expressão Gênica , Éteres Difenil Halogenados/administração & dosagem , Humanos , Íntrons/genética , Metabolismo dos Lipídeos , PPAR alfa/metabolismo , Receptor de Pregnano X/genética , Receptor de Pregnano X/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptor X Retinoide alfa/metabolismo
9.
Nature ; 579(7797): 123-129, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32103176

RESUMO

A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families that are known to be produced by the microbiome have a marked effect on the balance between health and disease1-9. Considering the diversity of the human microbiome (which numbers over 40,000 operational taxonomic units10), the effect of the microbiome on the chemistry of an entire animal remains underexplored. Here we use mass spectrometry informatics and data visualization approaches11-13 to provide an assessment of the effects of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free and specific-pathogen-free mice. We found that the microbiota affects the chemistry of all organs. This included the amino acid conjugations of host bile acids that were used to produce phenylalanocholic acid, tyrosocholic acid and leucocholic acid, which have not previously been characterized despite extensive research on bile-acid chemistry14. These bile-acid conjugates were also found in humans, and were enriched in patients with inflammatory bowel disease or cystic fibrosis. These compounds agonized the farnesoid X receptor in vitro, and mice gavaged with the compounds showed reduced expression of bile-acid synthesis genes in vivo. Further studies are required to confirm whether these compounds have a physiological role in the host, and whether they contribute to gut diseases that are associated with microbiome dysbiosis.


Assuntos
Ácidos e Sais Biliares/biossíntese , Ácidos e Sais Biliares/química , Metabolômica , Microbiota/fisiologia , Animais , Ácidos e Sais Biliares/metabolismo , Ácido Cólico/biossíntese , Ácido Cólico/química , Ácido Cólico/metabolismo , Fibrose Cística/genética , Fibrose Cística/metabolismo , Fibrose Cística/microbiologia , Vida Livre de Germes , Humanos , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/metabolismo , Doenças Inflamatórias Intestinais/microbiologia , Camundongos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo
10.
Cell Immunol ; 349: 104047, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32019673

RESUMO

The polarization of macrophages is critical to inflammation and tissue repair, with unbalanced macrophage polarization associated with critical dysfunctions of the immune system. Cytochrome P450 1A1 (CYP1A1) is a hydroxylase mainly controlled by the inflammation-limiting aryl hydrocarbon receptor (AhR), which plays a critical role in mycoplasma infection, oxidative stress injury, and cancer. Arginase-1 (Arg-1) is a surrogate for polarized alternative macrophages and is important to the production of nitric oxide (NO) by the modulation of arginine. In the present study, we found CYP1A1 to be upregulated in IL-4-stimulated mouse peritoneal macrophages (PMs) and human peripheral blood monocytes. Using CYP1A1-overexpressing RAW264.7 cells (CYP1A1/RAW) we found that CYP1A1 augmented Arg-1 expression by strengthening the activation of the JAK1/STAT6 signaling pathway in macrophages treated with IL-4. 15(S)-HETE, a metabolite of CYP1A1 hydroxylase, was elevated in IL-4-induced CYP1A1/RAW cells. Further, in macrophages, the loss-of-CYP1A1-hydroxylase activity was associated with reduced IL-4-induced Arg-1 expression due to impaired 15(S)-HETE generation. Of importance, CYP1A1 overexpressing macrophages reduced the inflammation associated with LPS-induced peritonitis. Taken together, these findings identified a novel signaling axis, CYP1A1-15(S)-HETE-JAK1-STAT6, that may be a promising target for the proper maintenance of macrophage polarization and may also be a means by which to treat immune-related disease due to macrophage dysfunction.


Assuntos
Arginase/biossíntese , Citocromo P-450 CYP1A1/fisiologia , Janus Quinase 1/antagonistas & inibidores , Macrófagos Peritoneais/efeitos dos fármacos , Peritonite/prevenção & controle , Fator de Transcrição STAT6/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Transferência Adotiva , Animais , Araquidonato 15-Lipoxigenase/fisiologia , Arginase/genética , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/genética , Endotoxinas/toxicidade , Humanos , Ácidos Hidroxieicosatetraenoicos/biossíntese , Ácidos Hidroxieicosatetraenoicos/genética , Ácidos Hidroxieicosatetraenoicos/farmacologia , Interleucina-4/farmacologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/transplante , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peritonite/induzido quimicamente , Células RAW 264.7 , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/biossíntese , Receptores Citoplasmáticos e Nucleares/genética , Células THP-1 , Regulação para Cima/efeitos dos fármacos
11.
Toxicology ; 433-434: 152394, 2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-32027962

RESUMO

Nongenotoxic chemicals can produce liver tumours in rats and mice by a mitogenic mode of action involving activation of the constitutive androstane receptor (CAR). The aim of this study was to evaluate the usefulness of cultured hepatocytes from normal (wild type; WT) and CAR knockout (KO) rats to screen compounds as potential activators of rat CAR and to validate this test system. Cultured hepatocytes from male Sprague-Dawley WT and CAR KO rats were treated with either 100 and 1000 µM sodium phenobarbital (NaPB), 3-100 µM fluquinconazole (FQZ), or 3-300 µM 3-(difluoromethyl)-1-methyl-N-(3´,4´,6-trifluoro[1,1´-biphenyl]-2-yl)-1H-pyrazole-4-carboxamide (TI1) for 96 h. Induction of cytochrome P450 (CYP) enzymes was monitored by measurement of 7-pentoxyresorufin O-depentylase (PROD), 7-benzyloxyresorufin O-debenzylase (BROD) and 7-benzyloxyquinoline O-debenzylase (BQ) activities. Hepatocytes undergoing replicative DNA synthesis (RDS) were labelled by adding 10 µM 5-bromo-2´-deoxyuridine to the culture medium for determination of the hepatocyte labelling index. The treatment of WT, but not of CAR KO, rat hepatocytes with NaPB, FQZ and TI1 increased hepatocyte RDS and induced CYP2B-dependent PROD activity. In contrast, all three compounds increased CYP2B/3A-dependent BROD and CYP3A-dependent BQ activities in both WT and CAR KO rat hepatocytes. Hepatocyte RDS was increased in both WT and CAR KO rat hepatocytes by treatment with 25 ng/ml epidermal growth factor as a positive control. Overall, these results demonstrate that the effects of three CAR activators on RDS and CYP2B enzyme induction are abolished in cultured CAR KO rat hepatocytes. As demonstrated by this validation study, the CAR KO hepatocyte model is a useful in vitro mechanistic tool for the rapid screening of chemicals as potential activators of rat CAR.


Assuntos
Hepatócitos/efeitos dos fármacos , Fenobarbital/farmacologia , Quinazolinonas/farmacologia , Receptores Citoplasmáticos e Nucleares/genética , Triazóis/farmacologia , Animais , Indutores das Enzimas do Citocromo P-450/administração & dosagem , Indutores das Enzimas do Citocromo P-450/farmacologia , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Sistema Enzimático do Citocromo P-450/metabolismo , DNA/metabolismo , Relação Dose-Resposta a Droga , Técnicas de Inativação de Genes , Masculino , Camundongos Knockout , Fenobarbital/administração & dosagem , Quinazolinonas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Triazóis/administração & dosagem
12.
Diabetes Res Clin Pract ; 161: 108033, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32006644

RESUMO

Previous studies have shown that FXR is involved in glycolipid metabolism, tissue inflammation and regeneration in organs such as the liver, intestines and kidneys. Although FXR has been reported in cardiac tissue, its function in diabetic cardiomyopathy has not been reported. Here, we successfully constructed a diabetic mouse model of FXR-/- and evaluated the effects of FXR knockout on cardiac function in mice by measuring various indicators. We demonstrated that blood glucose levels in diabetic mice are significantly elevated in the case of FXR knockout. Our findings from cardiac ultrasound and tissue HE staining supported that FXR knockout aggravates diabetic cardiomyopathy. Masson staining of myocardial tissue and quantitative detection of α-SMA by qPCR suggest that FXR knockout exacerbates cardiac fibrosis in diabetic cardiomyopathy. Combined with the results of Oil Red staining and quantitative detection of triglycerides in fresh tissue blocks, we hypothesized that FXR knockout aggravates diabetes-induced cardiac lipid accumulation. Altogether our results revealed a role of the FXR in the diabetic cardiomyopathy, suggesting a possible novel target for the treatment of diabetic cardiomyopathy.


Assuntos
Diabetes Mellitus Experimental/complicações , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/patologia , Receptores Citoplasmáticos e Nucleares/genética , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Cardiomiopatias Diabéticas/metabolismo , Progressão da Doença , Inflamação/complicações , Inflamação/genética , Inflamação/metabolismo , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Miocárdio/patologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Estreptozocina , Triglicerídeos/sangue
13.
BMC Cancer ; 20(1): 85, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005108

RESUMO

BACKGROUND: Interrogation of site-specific CpG methylation in circulating tumor DNAs (ctDNAs) has been employed in a number of studies for early detection of breast cancer (BrCa). In many of these studies, the markers were identified based on known biology of BrCa progression, and interrogated using methyl-specific PCR (MSP), a technique involving bisulfite conversion, PCR, and qPCR. METHODS: In this report, we are demonstrating the development of a novel assay (Multiplex Bisulfite PCR-LDR-qPCR) which can potentially offer improvements to MSP, by integrating additional steps such as ligase detection reaction (LDR), methylated CpG target enrichment, carryover protection (use of uracil DNA glycosylase), and minimization of primer-dimer formation (use of ribose primers and RNAseH2). The assay is designed to for breast cancer-specific CpG markers identified through integrated analyses of publicly available genome-wide methylation datasets for 31 types of primary tumors (including BrCa), as well as matching normal tissues, and peripheral blood. RESULTS: Our results indicate that the PCR-LDR-qPCR assay is capable of detecting ~ 30 methylated copies of each of 3 BrCa-specific CpG markers, when mixed with excess amount unmethylated CpG markers (~ 3000 copies each), which is a reasonable approximation of BrCa ctDNA overwhelmed with peripheral blood cell-free DNA (cfDNA) when isolated from patient plasma. The bioinformatically-identified CpG markers are located in promoter regions of NR5A2 and PRKCB, and a non-coding region of chromosome 1 (upstream of EFNA3). Additional bioinformatic analyses would reveal that these methylation markers are independent of patient race and age, and positively associated with signaling pathways associated with BrCa progression (such as those related to retinoid nuclear receptor, PTEN, p53, pRB, and p27). CONCLUSION: This report demonstrates the potential utilization of bisulfite PCR-LDR-qPCR assay, along with bioinformatically-driven biomarker discovery, in blood-based BrCa detection.


Assuntos
Biomarcadores Tumorais/sangue , Neoplasias da Mama/diagnóstico , Ácidos Nucleicos Livres/sangue , Metilação de DNA , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Ilhas de CpG , Feminino , Humanos , Células MCF-7 , Reação em Cadeia da Polimerase Multiplex , Proteína Quinase C beta/genética , Receptores Citoplasmáticos e Nucleares/genética
14.
Expert Opin Pharmacother ; 21(4): 399-408, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31957504

RESUMO

Introduction: Despite unprecedented advances in the treatment of multiple myeloma (MM), almost all patients develop a disease that is resistant to the five most commonly used and active anti-MM agents. The prognosis for this patient population is particularly poor resulting in an unmet need for additional therapeutic options. Exportin-1 (XPO-1) is a major nuclear export protein of macromolecular cargo frequently overexpressed in MM. Selinexor is a first-in-class, oral Selective-Inhibitor-of-Nuclear-Export (SINE) compound that impedes XPO-1. Based on results of the STORM-trial, selinexor in combination with dexamethasone was granted accelerated FDA approval for patients with penta-refractory MM in July 2019.Areas covered: This article summarizes our up-to-date knowledge on the pathophysiologic role of XPO-1 in MM. Furthermore, it reviews the most recent clinical data on selinexor in combination with dexamethasone and other anti-MM agents; and discusses its safety profile, management strategies; and potential future developments.Expert opinion: Selinexor represents a next-generation-novel agent with an innovative mechanism of action that marks a significant advance in the treatment of heavily pretreated MM patients. Ongoing studies investigate its therapeutic potential also in earlier lines of therapy. Additional data is needed to confirm that selinexor and other SINE compounds are a valuable addition to our current therapeutic armamentarium.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Hidrazinas/uso terapêutico , Carioferinas/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Triazóis/uso terapêutico , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ensaios Clínicos como Assunto , Dexametasona/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Humanos , Hidrazinas/administração & dosagem , Hidrazinas/efeitos adversos , Hidrazinas/farmacocinética , Carioferinas/genética , Mieloma Múltiplo/metabolismo , Prognóstico , Receptores Citoplasmáticos e Nucleares/genética , Triazóis/administração & dosagem , Triazóis/efeitos adversos , Triazóis/farmacocinética
15.
J Toxicol Sci ; 45(1): 25-35, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31932555

RESUMO

In this study, we found that nuclear receptors FXR and LXR (originally characterized as regulatory factors involved in cholesterol/bile acid homeostasis) regulate the expression of Oct3/4, a marker for cell differentiation, in both normal renal-derived cell line HK-2 and renal adenocarcinoma cell line ACHN. Down-regulation of Oct3/4 expression by activating FXR and LXR occurs only in normal renal cell-derived HK-2 cells. We also found that the RNA-binding protein, ELAVL2, oppositely regulates Oct3/4 expressions in HK-2 and ACHN cells. Moreover, we revealed that LXR-alpha and LXR-beta regulate each other's expression. Although an LXR-beta-specific agonist is assumed to be the basis for an anti-arteriosclerotic drug that only stimulates reverse cholesterol transport, our findings show that the development of such an anti-arteriosclerotic drug would require further elucidation of the complex mechanism of LXR-alpha and LXR-beta regulation.


Assuntos
Adenocarcinoma , Regulação da Expressão Gênica , Neoplasias Renais , Rim/citologia , Receptores X do Fígado/genética , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Arteriosclerose/tratamento farmacológico , Linhagem Celular , Colesterol/metabolismo , Regulação para Baixo , Descoberta de Drogas , Proteína Semelhante a ELAV 2/genética , Humanos
16.
Mol Cells ; 43(1): 34-47, 2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-31896234

RESUMO

The circadian clock regulates various physiological processes, including bone metabolism. The nuclear receptors Reverbs, comprising Rev-erbα and Rev-erbß, play a key role as transcriptional regulators of the circadian clock. In this study, we demonstrate that Rev-erbs negatively regulate differentiation of osteoclasts and osteoblasts. The knockdown of Rev-erbα in osteoclast precursor cells enhanced receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation, as well as expression of nuclear factor of activated T cells 1 (NFATc1), osteoclast-associated receptor (OSCAR), and tartrate-resistant acid phosphatase (TRAP). The overexpression of Rev-erbα leads to attenuation of the NFATc1 expression via inhibition of recruitment of c-Fos to the NFATc1 promoter. The overexpression of Rev-erbα in osteoblast precursors attenuated the expression of osteoblast marker genes including Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OC). Rev-erbα interfered with the recruitment of Runx2 to the promoter region of the target genes. Conversely, knockdown of Reverbα in the osteoblast precursors enhanced the osteoblast differentiation and function. In addition, Rev-erbα negatively regulated osteoclast and osteoblast differentiation by suppressing the p38 MAPK pathway. Furthermore, intraperitoneal administration of GSK4112, a Rev-erb agonist, protects RANKL-induced bone loss via inhibition of osteoclast differentiation in vivo . Taken together, our results demonstrate a molecular mechanism of Rev-erbs in the bone remodeling, and provide a molecular basis for a potential therapeutic target for treatment of bone disease characterized by excessive bone resorption.


Assuntos
Reabsorção Óssea/metabolismo , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Osteoblastos/fisiologia , Osteoclastos/fisiologia , Osteogênese/genética , Animais , Remodelação Óssea , Reabsorção Óssea/genética , Diferenciação Celular , Células Cultivadas , Relógios Circadianos , Modelos Animais de Doenças , Mutação com Ganho de Função/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , RNA Interferente Pequeno/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
17.
FASEB J ; 34(2): 3367-3378, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31919912

RESUMO

Ppardδ, one of the lipid-activated nuclear receptor expressed in many cell types to activate gene transcription, also regulates cellular functions other than lipid metabolism. The mechanism regulating the function of antigen-presenting cells during the development of atherosclerosis is not fully understood. Here we aimed to study the involvement of PPARδ in CD11c+ cells in atherosclerosis. We used the Cre-loxP approach to make conditional deletion of Ppard in CD11c+ cells in mice on Apoe-/- background, which were fed with high cholesterol diet to develop atherosclerosis. Ppard deficiency in CD11c+ cells attenuated atherosclerotic plaque formation and infiltration of myeloid-derived dendritic cells (DCs) and T lymphocytes. Reduced lesion was accompanied by reduced activation of dendritic cells, and also a reduction of activation and differentiation of T cells to Th1 cells. In addition, DC migration to lymph node was also attenuated with Ppard deletion. In bone marrow-derived DCs, Ppard deficiency reduced palmitic acid-induced upregulation of co-stimulatory molecules and pro-inflammatory cytokine IL12 and TNFα. Our results indicated PPARδ activation by fatty acid resulted in the activation of myeloid DCs and subsequent polarization of T lymphocytes, which contributed to atherosclerosis in Apoe-/- mice. These findings also reveal the potential regulatory role of PPARδ in antigen presentation to orchestrate the immune responses during atherosclerosis.


Assuntos
Aterosclerose/metabolismo , Células Dendríticas/metabolismo , Deleção de Genes , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Antígenos CD11/genética , Antígenos CD11/metabolismo , Células Cultivadas , Interleucina-12/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptores Citoplasmáticos e Nucleares/genética , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
18.
Biochem Biophys Res Commun ; 523(3): 766-772, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-31948764

RESUMO

Long noncoding RNA small nucleolus RNA host gene 14 (SNHG14) has been shown to exert oncogenic functions in seceral cancers, but its role in osteosarcoma still unclear. In this present study, we found that SNHG14 was significantly upregulated in osteosarcoma tissues and cell lines. Knockdown SNHG14 expression significantly inhibited osteosarcoma cell proliferation through inducing apoptosis. Further functional assays revealed that SNHG14 knockdown dramatically suppressed cell migration and invasion. Bioinformatics analysis and luciferase assays identified that microRNA-433-3p (miR-433-3p) was a direct target of SNHG14, and directly targeted F-box only protein 22 (FBXO22). Mechanistic analysis demonstrated that SNHG14 acted as a ceRNA in modulating osteosarcoma proliferation, migration and invasion by decoying miR-433-3p to upregulate FBXO22 expression. We also observed that knockdown of FBXO22 and SNHG14 and overexpression of miR-433-3p both dramatically suppressed osteosarcoma cell proliferation, migration and invasion, but induced cell apoptosis. Moreover, the suppresive effect of SNHG14 knockdown on osteosarcoma cell proliferation, invasion and migration was attenuated by miR-433-3p inhibitor. Our findings demonstrated that SNHG14 promoted osteosarcoma progression by acting as a ceRNA for miR-433-3p to regulate FBXO22 expression, suggesting that SNHG14 may serve as a potential therapeutic target in osteosarcoma patients.


Assuntos
Neoplasias Ósseas/genética , Proteínas F-Box/genética , MicroRNAs/genética , Osteossarcoma/genética , RNA Longo não Codificante/genética , Receptores Citoplasmáticos e Nucleares/genética , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Osteossarcoma/patologia
19.
J Biol Chem ; 295(9): 2850-2865, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31911440

RESUMO

Cholesterol synthesis is a tightly regulated process, both transcriptionally and post-translationally. Transcriptional control of cholesterol synthesis is relatively well-understood. However, of the ∼20 enzymes in cholesterol biosynthesis, post-translational regulation has only been examined for a small number. Three of the four sterol reductases in cholesterol production, 7-dehydrocholesterol reductase (DHCR7), 14-dehydrocholesterol reductase (DHCR14), and lamin-B receptor (LBR), share evolutionary ties with a high level of sequence homology and predicted structural homology. DHCR14 and LBR uniquely share the same Δ-14 reductase activity in cholesterol biosynthesis, yet little is known about their post-translational regulation. We have previously identified specific modes of post-translational control of DHCR7, but it is unknown whether these regulatory mechanisms are shared by DHCR14 and LBR. Using CHO-7 cells stably expressing epitope-tagged DHCR14 or LBR, we investigated the post-translational regulation of these enzymes. We found that DHCR14 and LBR undergo differential post-translational regulation, with DHCR14 being rapidly turned over, triggered by cholesterol and other sterol intermediates, whereas LBR remained stable. DHCR14 is degraded via the ubiquitin-proteasome system, and we identified several DHCR14 and DHCR7 putative interaction partners, including a number of E3 ligases that modulate DHCR14 levels. Interestingly, we found that gene expression across an array of human tissues showed a negative relationship between the C14-sterol reductases; one enzyme or the other tends to be predominantly expressed in each tissue. Overall, our findings indicate that whereas LBR tends to be the constitutively active C14-sterol reductase, DHCR14 levels are tunable, responding to the local cellular demands for cholesterol.


Assuntos
Colesterol/biossíntese , Regulação da Expressão Gênica , Oxirredutases/genética , Processamento de Proteína Pós-Traducional , Receptores Citoplasmáticos e Nucleares/genética , Animais , Células CHO , Cricetulus , Humanos , Especificidade de Órgãos , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Estabilidade Proteica , Ubiquitina-Proteína Ligases/metabolismo
20.
Comput Biol Chem ; 84: 107163, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31767507

RESUMO

The present study was to illustrate the agonistic property of arjungenin and arjunic acid towards farnesoid X receptor protein (FXR).The pharmacokinetic properties like molecular interactions, absorption, distribution, metabolism, elimination and toxicity (ADMET) of the ligands were checked through in-silico studies. Protein-ligand docking was carried out using autodock software. Molecular docking analysis confirmed strong binding energy and interaction of arjungenin and arjunic acid with the target protein and the ADMET profiles identified for both compounds were promising.Further in vitro studies were performed in 3T3-L1 adipocyte to verify the agonistic property of arjungenin and arjunic acid. Oil red O staining was done to check differentiation induction. Adiponectin, leptin, triglycerides and total cholesterol levels were quantified. The mRNA expression of FXR, Cyp7a1, PPAR-γ and SREBP-1c were quantified using fluorescent real-time PCR. Cytotoxicity assay was confirmed that up to 150 µM concentration there is no significant cell death on treatment with arjunic acid and arjungenin. Treatment with arjungenin and arjunic acid confirms increased differentiation of the cells with significant (P < 0.05) increase in adiponectin (118.07% and 132.92%) and leptin (133.52% and 149.74%) protein levels compared to the negative control group. After treatment with arjungenin and arjunic acid in 3T3-L1 preadipocytes the mRNA expression of FXR, PPAR-γ and SREBP-1c were significantly (P < 0.01) increased and cyp7a1 was significantly (P < 0.01) decreased when compared with the negative control group. Overall, our results suggest that arjungenin and arjunic acid acts as an FXR agonist and may be useful for rational therapeutic strategies as a novel drug to treat cholesterol mediated metabolic syndrome and insulin resistance.


Assuntos
Receptores Citoplasmáticos e Nucleares/agonistas , Triterpenos/farmacologia , Células 3T3-L1 , Adiponectina/metabolismo , Animais , Colesterol 7-alfa-Hidroxilase/genética , Colesterol 7-alfa-Hidroxilase/metabolismo , Expressão Gênica/efeitos dos fármacos , Leptina/metabolismo , Camundongos , Simulação de Acoplamento Molecular , PPAR gama/genética , PPAR gama/metabolismo , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triterpenos/metabolismo , Triterpenos/farmacocinética , Triterpenos/toxicidade
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