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1.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 36(9): 905-909, 2019 Sep 10.
Artigo em Chinês | MEDLINE | ID: mdl-31515787

RESUMO

OBJECTIVE: To detect mutation of LBR gene in a pedigree affected with Pelger-Huёt anomaly (PHA) and to explore its clinical characteristics. METHODS: Genomic DNA was extracted from the pedigree and healthy controls. The 14 exons of the LBR gene were subjected to PCR amplification and Sanger sequencing. Suspected mutations were verified in other family members and 100 healthy controls. Polyphen-2 and SIFT software were used to predict the effect of the mutation, and Swiss-model software was used to simulate the protein structure. RESULTS: Three patients were found to carry a c.893G>A mutation in exon 8 of the LBR gene, which resulted in substitution of the 298th amino acid residue glycine by glutamic acid (p.Gly298Glu). The same mutation was not found in healthy family members and 100 healthy controls. The mutation was predicted to be damaging. Bioinformatic simulation showed the mutation has altered the 3D structure of the LBR protein. CONCLUSION: The c.893G>A (p.Gly298Glu) mutation in the LBR gene probably underlies the PHA in this pedigree and has enriched the spectrum of LBR gene mutations.


Assuntos
Anomalia de Pelger-Huët/genética , Receptores Citoplasmáticos e Nucleares/genética , Estudos de Casos e Controles , Análise Mutacional de DNA , Éxons , Humanos , Mutação , Linhagem , Reação em Cadeia da Polimerase
2.
Chem Biol Interact ; 311: 108794, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31421115

RESUMO

Acanthoic acid (AA) is a pimaradiene diterpene isolated from Acanthopanax koreanum Nakai (Araliaceae), with anti-inflammatory and hepatic-protective effects. The present study intended to reveal the effect and mechanism of AA on nonalcoholic fatty liver disease (NAFLD) associated with lipid accumulation by activating Farnesoid X receptor (FXR) and liver X receptors (LXRs) signaling. C57BL/6 mice were received a modified Lieber-DeCarli diet with 71% high-fat (L-D) and treated with AA (20 and 40 mg/kg) or equal volume of saline for 12 weeks. The regulation of AA on lipid accumulation was also detected in pro-steatotic stimulated AML12 cells with palmitic acid (PA). When L-D diet-fed mice were treated with AA, loss in body weight, liver index, and liver lipid droplet were observed along with reduced triglyceride (TG) and serum transaminase. Furthermore, AA decreased sterol regulatory element binding protein 1 (SREBP-1) and target genes expression, regulated PPARα and PPARγ expressions, ameliorated hepatic fibrosis markers, enhanced hepatic FXR and LXR, and regulated AMPK-LKB1 and SIRT1 signaling pathway. Moreover, AA attenuated lipid accumulation via FXR and LXR activation in steatotic AML-12 cells, which was confirmed by guggulsterones (FXR antagonist) or GW3965 (LXR agonist). Activation of FXR and LXR signaling caused by AA might increase AMPK-SIRT1 signaling and then contribute to modulating lipid accumulation and fatty acid synthesis, which suggested that activated FXR-LXR axis by AA represented an effective strategy for relieving NAFLD.


Assuntos
Diterpenos/farmacologia , Lipogênese/efeitos dos fármacos , Receptores X do Fígado/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Peso Corporal/efeitos dos fármacos , Linhagem Celular , Dieta Hiperlipídica , Diterpenos/química , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores X do Fígado/agonistas , Receptores X do Fígado/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , PPAR alfa/genética , PPAR alfa/metabolismo , Ácido Palmítico/farmacologia , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Transdução de Sinais/efeitos dos fármacos , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/sangue
3.
Genes Dev ; 33(15-16): 1083-1094, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31296559

RESUMO

The orphan nuclear receptor SHP (small heterodimer partner) is a well-known transcriptional corepressor of bile acid and lipid metabolism in the liver; however, its function in other tissues is poorly understood. Here, we report an unexpected role for SHP in the exocrine pancreas as a modulator of the endoplasmic reticulum (ER) stress response. SHP expression is induced in acinar cells in response to ER stress and regulates the protein stability of the spliced form of X-box-binding protein 1 (XBP1s), a key mediator of ER stress response. Loss of SHP reduces XBP1s protein level and transcriptional activity, which in turn attenuates the ER stress response during the fasting-feeding cycle. Consequently, SHP-deficient mice also are more susceptible to cerulein-induced pancreatitis. Mechanistically, we show that SHP physically interacts with the transactivation domain of XBP1s, thereby inhibiting the polyubiquitination and degradation of XBP1s by the Cullin3-SPOP (speckle-type POZ protein) E3 ligase complex. Together, our data implicate SHP in governing ER homeostasis and identify a novel posttranslational regulatory mechanism for the key ER stress response effector XBP1.


Assuntos
Estresse do Retículo Endoplasmático/genética , Proteólise , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Células Acinares/metabolismo , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas Exócrino/metabolismo , Pancreatite/genética , Processamento de Proteína , Estabilidade Proteica , Receptores Citoplasmáticos e Nucleares/deficiência , Receptores Citoplasmáticos e Nucleares/genética , Ubiquitinação/genética
4.
J Agric Food Chem ; 67(32): 8868-8874, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31319027

RESUMO

Chenodeoxycholic acid (CDCA), a primary bile acid, has been demonstrated to play important roles as a signaling molecule in various physiology functions. However, the role of CDCA in regulating intestinal barrier function remains largely unknown. This study aimed to investigate the effects of CDCA on the lipopolysaccharide (LPS)-impaired intestinal epithelial barrier function and explore the underlying mechanisms. In IPEC-J2 cells, CDCA reversed the LPS-induced increase in transepithelial electrical resistance and decrease in tight junction protein expression. In addition, we found that farnesoid X receptor (FXR) but not Takeda G-protein receptor 5 was responsible for the CDCA-improved epithelial barrier function impaired by LPS. Furthermore, CDCA blocked LPS-induced activation of the myosin light chain kinase (MLCK) pathway in a FXR-dependent manner and elicited similar effects to MLCK inhibition. In mice, CDCA supplementation restored LPS-induced elevation of intestinal permeability and MLCK expression and reduction of tight junction protein expression, thus alleviating LPS-induced intestinal barrier impairment. In conclusion, CDCA protected against the LPS-induced impairment of the intestinal epithelial barrier function via the FXR-MLCK pathway.


Assuntos
Ácido Quenodesoxicólico/administração & dosagem , Enteropatias/tratamento farmacológico , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Lipopolissacarídeos/efeitos adversos , Quinase de Cadeia Leve de Miosina/metabolismo , Substâncias Protetoras/administração & dosagem , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Células CACO-2 , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Enteropatias/induzido quimicamente , Enteropatias/genética , Enteropatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Quinase de Cadeia Leve de Miosina/genética , Receptores Citoplasmáticos e Nucleares/genética , Transdução de Sinais/efeitos dos fármacos , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/genética , Junções Íntimas/metabolismo
5.
Anticancer Res ; 39(7): 3601-3608, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262885

RESUMO

BACKGROUND/AIM: Nuclear receptors regulate the expression of cellular transporters, which may be contributing factors for cisplatin (CDDP) resistance. This study aimed to clarify whether nuclear receptor ligands could be potentially used as drugs to overcome CDDP resistance. MATERIALS AND METHODS: Caspase-3 activity was measured using a fluorogenic substrate. mRNA levels were determined using real-time polymerase chain reaction. RESULTS: Pregnane X receptor (PXR) showed an expression level change dependent on caspase-3 activation by CDDP in HepG2. Rifampicin, a PXR agonist, reduced the accumulation of CDDP and suppressed growth inhibition and caspase-3 activation in HepG2 after CDDP exposure. Leflunomide, a PXR antagonist, significantly enhanced caspase-3 activation by CDDP in HepG2 and CDDP-resistant HepG2/R. CONCLUSION: These results suggest that PXR can modify the antitumor activity of CDDP, presumably through regulating the expression of transporters, which control intracellular CDDP concentration. Thus, PXR antagonists can be further investigated as potential drugs capable of overcoming CDDP resistance.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/farmacologia , Receptores Citoplasmáticos e Nucleares/genética , Caspase 3/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Hep G2 , Humanos , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos
6.
Nat Commun ; 10(1): 2915, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266946

RESUMO

The bile acid-sensing transcription factor farnesoid X receptor (FXR) regulates multiple metabolic processes. Modulation of FXR is desired to overcome several metabolic pathologies but pharmacological administration of full FXR agonists has been plagued by mechanism-based side effects. We have developed a modulator that partially activates FXR in vitro and in mice. Here we report the elucidation of the molecular mechanism that drives partial FXR activation by crystallography- and NMR-based structural biology. Natural and synthetic FXR agonists stabilize formation of an extended helix α11 and the α11-α12 loop upon binding. This strengthens a network of hydrogen bonds, repositions helix α12 and enables co-activator recruitment. Partial agonism in contrast is conferred by a kink in helix α11 that destabilizes the α11-α12 loop, a critical determinant for helix α12 orientation. Thereby, the synthetic partial agonist induces conformational states, capable of recruiting both co-repressors and co-activators leading to an equilibrium of co-activator and co-repressor binding.


Assuntos
Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/química , Animais , Linhagem Celular , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Humanos , Ligações de Hidrogênio , Ligantes , Fígado/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Conformação Proteica em alfa-Hélice , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo
7.
Dokl Biochem Biophys ; 485(1): 138-140, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31201635

RESUMO

To study the mechanisms of transcriptional regulation, a convenient experimental approach is to use the artificial chimeric constructs carrying the regulatory elements of interest. In the present work, we describe the creation and characterization of a novel genetic construct that makes it possible to study the transcriptional regulation of the early-late gene of the ecdysone cascade. Using the data of genome-wide experiments, we have isolated the main regulatory region of the hr4 gene, which was successfully used to create a chimeric reporter construct expressing a fluorescent protein upon the treatment with the ecdysone hormone. This reporter system can be used to study the mechanisms of the ecdysone response, both in cell culture and in tissues, at various stages of the Drosophila development.


Assuntos
Proteínas de Drosophila , Ecdisona/metabolismo , Genes Reporter , Receptores Citoplasmáticos e Nucleares , Transcrição Genética , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Estudo de Associação Genômica Ampla , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo
8.
Toxicol Lett ; 313: 1-10, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31170421

RESUMO

The constitutive androstane receptor(CAR) activation is connected with mitogenic effects leading to liver hyperplasia and tumorigenesis in rodents. CAR activators, including phenobarbital, are considered rodent non-genotoxic carcinogens. Recently, trans-3,4,5,4´-tetramethoxystilbene(TMS), a potential anticancer drug (DMU-212), have been shown to alleviate N-nitrosodiethylamine/phenobarbital-induced liver carcinogenesis. We studied whether TMS inhibits mouse Car to protect from the PB-induced tumorigenesis. Unexpectedly, we identified TMS as a murine CAR agonist in reporter gene experiments, in mouse hepatocytes, and in C57BL/6 mice in vivo. TMS up-regulated Car target genes Cyp2b10, Cyp2c29 and Cyp2c55 mRNAs, but down-regulated expression of genes involved in gluconeogenesis and lipogenesis. TMS did not change or down-regulate genes involved in liver proliferation or apoptosis such as Mki67, Foxm1, Myc, Mcl1, Pcna, Bcl2, or Mdm2, which were up-regulated by another Car ligand TCPOBOP. TMS did not increase liver weight and had no significant effect on Ki67 and Pcna labeling indices in mouse liver in vivo. In murine hepatic AML12 cells, we confirmed a Car-independent proapoptotic effect of TMS. We conclude that TMS is a Car ligand with limited effects on hepatocyte proliferation, likely due to promoting apoptosis in mouse hepatic cells, while controlling Car target genes involved in xenobiotic and endobiotic metabolism.


Assuntos
Anticarcinógenos/farmacologia , Proliferação de Células/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Neoplasias Hepáticas/prevenção & controle , Fígado/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/agonistas , Estilbenos/farmacologia , Animais , Anticarcinógenos/metabolismo , Apoptose/efeitos dos fármacos , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Sítios de Ligação , Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Gluconeogênese/efeitos dos fármacos , Gluconeogênese/genética , Células Hep G2 , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Fígado/metabolismo , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Ligação Proteica , Piridinas/farmacologia , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Estilbenos/metabolismo
9.
Environ Pollut ; 246: 945-954, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31159144

RESUMO

Polychlorinated biphenyls (PCBs) are persistent organic pollutants and hazardous to human health. Aflatoxin B1 (AFB1) is a strong carcinogen dependent on activation by cytochrome P450 (CYP) 1A2 and 3A4. Humans in some regions may be exposed to both PCBs and AFB1. Since PCBs are CYP inducers, we were interested in their combined genotoxicity. In this study, the effects of non-coplanar 2,3,3'-tri- (PCB 20), 2,2'5,5'-tetra- (PCB 52), 2,3,3',4'-tetrachlorobiphenyl (PCB 56), and coplanar 3,3',4,4',5-pentachlorobiphenyl (PCB 126) on protein levels of CYP1A1, 1A2, and 3A4, and nuclear receptors AhR, CAR and PXR in a human hepatocyte (L-02) line were investigated. Moreover, the combined effects of each PCB and AFB1 for induction of micronuclei and double-strand DNA breaks (indicated by an elevation of γ-H2AX) were analyzed. The results indicated that PCBs 20, 52 and 56 reduced the expression of AhR, while elevated that of CAR and PXR, with thresholds at low micromolar concentrations. However, they were less potent than PCB 126, which was active at sub-nanomolar levels. Overexpression of human splice variant CAR 3 in the cells increased CYP1A2 and 3A4 levels, which were further enhanced by each non-coplanar PCB, suggesting a role of CAR in modulating CYPs. Pretreatment of cells with each test PCB potentiated both micronuclei formation and DNA damage induced by AFB1. This study suggests that both non-coplanar and coplanar PCBs may enhance the genotoxicity of AFB1, through acting on various nuclear receptors; the potentiation of AFB1 genotoxicity by PCBs and the potential health implications may deserve concerns and further investigation.


Assuntos
Aflatoxina B1/toxicidade , Carcinógenos/toxicidade , Sistema Enzimático do Citocromo P-450/genética , Poluentes Ambientais/toxicidade , Bifenilos Policlorados/toxicidade , Linhagem Celular , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP3A/genética , Poluentes Ambientais/química , Hepatócitos/efeitos dos fármacos , Humanos , Bifenilos Policlorados/química , Receptores Citoplasmáticos e Nucleares/genética
10.
Int J Mol Sci ; 20(10)2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096545

RESUMO

Acute lymphoblastic leukemia is the most common type of childhood cancer worldwide. Mexico City has one of the highest incidences and mortality rates of this cancer. It has previously been recognized that chromosomal translocations are important in cancer etiology. Specific fusion genes have been considered as important treatment targets in childhood acute lymphoblastic leukemia (ALL). The present research aimed at the identification and characterization of novel fusion genes with potential clinical implications in Mexican children with acute lymphoblastic leukemia. The RNA-sequencing approach was used. Four fusion genes not previously reported were identified: CREBBP-SRGAP2B, DNAH14-IKZF1, ETV6-SNUPN, ETV6-NUFIP1. Although a fusion gene is not sufficient to cause leukemia, it could be involved in the pathogenesis of the disease. Notably, these new translocations were found in genes encoding for hematopoietic transcription factors which are known to play an important role in leukemogenesis and disease prognosis such as IKZF1, CREBBP, and ETV6. In addition, they may have an impact on the prognosis of Mexican pediatric patients with ALL, with the potential to be included in the current risk stratification schemes or used as therapeutic targets.


Assuntos
Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Translocação Genética/genética , Adolescente , Adulto , Proteína de Ligação a CREB/genética , Criança , Pré-Escolar , Dineínas/genética , Feminino , Proteínas Ativadoras de GTPase/genética , Regulação Neoplásica da Expressão Gênica , Rearranjo Gênico , Humanos , Fator de Transcrição Ikaros/genética , Lactente , Masculino , México , Proteínas Nucleares/genética , Prognóstico , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas de Ligação ao Cap de RNA/genética , Proteínas de Ligação a RNA/genética , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Repressoras/genética , Adulto Jovem
11.
Sci Total Environ ; 677: 626-636, 2019 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-31071665

RESUMO

Organic pollutants associated with diesel exhaust particles (DEP), such as polycyclic aromatic hydrocarbons (PAHs) and their derivatives, may negatively impact human health. However, a comprehensive overview of their effects on endocrine nuclear receptor activities is still missing. Here, we evaluated the effects of extracts and chromatographic fractions (fractionated according to increasing polarity) of two standard reference materials derived from distinct types of diesel engines (SRM 2975, SRM 1650b), on activation of androgen receptor (AR), estrogen receptor alpha (ERα), peroxisome proliferator-activated receptor γ (PPARγ), glucocorticoid receptor (GR) and thyroid receptor α (TRα), using human cell-based reporter gene assays. Neither DEP standard modulated AR or GR activities. Crude extracts and fractions of SRM 1650b and SRM 2975 suppressed ERα-mediated activity in the ER-CALUX™ assay; however, this effect could be partly linked to their cytotoxicity in this cell line. We observed that only SRM 2975 extract and its fractions were partial PPARγ inducers, while SRM 1650b extract was not active towards this receptor. Importantly, we found that both extracts and polar fractions of SRM activated TRα and significantly potentiated the activity of endogenous TRα ligand, triiodothyronine. Based on a detailed chemical analysis of both extracts and their polar fractions, we identified several oxygenated PAH derivatives, that were present at relatively high levels in the analyzed DEP standards, including 3-nitrobenzanthrone (3-NBA), anthracene-9,10-dione, phenanthrene-9,10-dione, 9H-fluoren-9-one or benzo[a]anthracene-7,12-dione, to activate TRα activity. Nevertheless, these compounds provided only a minor contribution to the overall TRα activity identified in polar fractions. This suggests that yet unidentified polar polyaromatic compounds associated with DEP may, apart from their known impact on the aryl hydrocarbon receptor or steroid signaling, deregulate activities of additional nuclear receptors, in particular of TRα. This illustrates the need to better characterize endocrine disrupting activities of DEP.


Assuntos
Poluentes Atmosféricos/efeitos adversos , Material Particulado/efeitos adversos , Hidrocarbonetos Policíclicos Aromáticos/efeitos adversos , Receptores Citoplasmáticos e Nucleares/genética , Emissões de Veículos , Linhagem Celular , Humanos , Receptores Citoplasmáticos e Nucleares/metabolismo
12.
Biomed Res Int ; 2019: 8973076, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31058195

RESUMO

Ovaries represent one of the primary steroidogenic organs, producing estrogen and progesterone under the regulation of gonadotropins during the estrous cycle. Gonadotropins fluctuate the expression of various steroidogenesis-related genes, such as those encoding steroidogenic enzymes, cholesterol deliverer, and electronic transporter. Steroidogenic factor-1 (SF-1)/adrenal 4-binding protein (Ad4BP)/NR5A1 and liver receptor homolog-1 (LRH-1) play important roles in these phenomena via transcriptional regulation. With the aid of cAMP, SF-1/Ad4BP and LRH-1 can induce the differentiation of stem cells into steroidogenic cells. This model is a useful tool for studying the molecular mechanisms of steroidogenesis. In this article, we will provide insight into the transcriptional regulation of steroidogenesis-related genes in ovaries that are revealed from stem cell-derived steroidogenic cells. Using the cells derived from the model, novel SF-1/Ad4BP- and LRH-1-regulated genes were identified by combined DNA microarray and promoter tiling array analyses. The interaction of SF-1/Ad4BP and LRH-1 with transcriptional regulators in the regulation of ovarian steroidogenesis was also revealed.


Assuntos
Ovário/crescimento & desenvolvimento , Receptores Citoplasmáticos e Nucleares/genética , Fator Esteroidogênico 1/genética , Transcrição Genética , Diferenciação Celular/genética , Feminino , Regulação da Expressão Gênica/genética , Humanos , Ovário/metabolismo , Regiões Promotoras Genéticas , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/genética
13.
MBio ; 10(3)2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088931

RESUMO

The abnormal proliferation of cancer cells is driven by deregulated oncogenes or tumor suppressors, among which the cancer-vulnerable genes are attractive therapeutic targets. Targeting mislocalization of oncogenes and tumor suppressors resulting from aberrant nuclear export is effective for inhibiting growth transformation of cancer cells. We performed a clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) screening in a unique model of matched primary and oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV)-transformed cells and identified genes that were growth promoting and growth suppressive for both types of cells, among which exportin XPO1 was demonstrated to be critical for the survival of transformed cells. Using XPO1 inhibitor KPT-8602 and by small interfering RNA (siRNA) knockdown, we confirmed the essential role of XPO1 in cell proliferation and growth transformation of KSHV-transformed cells and in cell lines of other cancers, including gastric cancer and liver cancer. XPO1 inhibition induced cell cycle arrest through p53 activation, but the mechanisms of p53 activation differed among the different types of cancer cells. p53 activation depended on the formation of promyelocytic leukemia (PML) nuclear bodies in gastric cancer and liver cancer cells. Mechanistically, XPO1 inhibition induced relocalization of autophagy adaptor protein p62 (SQSTM1), recruiting p53 for activation in PML nuclear bodies. Taken the data together, we have identified novel growth-promoting and growth-suppressive genes of primary and cancer cells and have demonstrated that XPO1 is a vulnerable target of cancer cells. XPO1 inhibition induces cell arrest through a novel PML- and p62-dependent mechanism of p53 activation in some types of cancer cells.IMPORTANCE Using a model of oncogenic virus KSHV-driven cellular transformation of primary cells, we have performed a genome-wide CRISPR-Cas9 screening to identify vulnerable genes of cancer cells. This screening is unique in that this virus-induced oncogenesis model does not depend on any cellular genetic alterations and has matched primary and KSHV-transformed cells, which are not available for similar screenings in other types of cancer. We have identified genes that are both growth promoting and growth suppressive in primary and transformed cells, some of which could represent novel proto-oncogenes and tumor suppressors. In particular, we have demonstrated that the exportin XPO1 is a critical factor for the survival of transformed cells. Using a XPO1 inhibitor (KPT-8602) and siRNA-mediated knockdown, we have confirmed the essential role of XPO1 in cell proliferation and in growth transformation of KSHV-transformed cells, as well as of gastric and liver cancer cells. XPO1 inhibition induces cell cycle arrest by activating p53, but the mechanisms of p53 activation differed among different types of cancer cells. p53 activation is dependent on the formation of PML nuclear bodies in gastric and liver cancer cells. Mechanistically, XPO1 inhibition induces relocalization of autophagy adaptor protein p62 (SQSTM1), recruiting p53 for activation in PML nuclear bodies. These results illustrate that XPO1 is a vulnerable target of cancer cells and reveal a novel mechanism for blocking cancer cell proliferation by XPO1 inhibition as well as a novel PML- and p62-mediated mechanism of p53 activation in some types of cancer cells.


Assuntos
Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Herpesvirus Humano 8/patogenicidade , Carioferinas/genética , Receptores Citoplasmáticos e Nucleares/genética , Sistemas CRISPR-Cas , Pontos de Checagem do Ciclo Celular , Detecção Precoce de Câncer , Genes p53 , Humanos , Carioferinas/antagonistas & inibidores , Leucemia Promielocítica Aguda , Neoplasias Hepáticas , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Proteína Sequestossoma-1/genética , Neoplasias Gástricas , Células Tumorais Cultivadas
14.
Int J Mol Sci ; 20(9)2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31083458

RESUMO

To appraise how evolutionary processes, such as gene duplication and loss, influence an organism's xenobiotic sensitivity is a critical question in toxicology. Of particular importance are gene families involved in the mediation of detoxification responses, such as members of the nuclear receptor subfamily 1 group I (NR1I), the pregnane X receptor (PXR), and the constitutive androstane receptor (CAR). While documented in multiple vertebrate genomes, PXR and CAR display an intriguing gene distribution. PXR is absent in birds and reptiles, while CAR shows a tetrapod-specific occurrence. More elusive is the presence of PXR and CAR gene orthologs in early branching and ecologically-important Chondrichthyes (chimaeras, sharks and rays). Therefore, we investigated various genome projects and use them to provide the first identification and functional characterization of a Chondrichthyan PXR from the chimaera elephant shark (Callorhinchus milii, Holocephali). Additionally, we substantiate the targeted PXR gene loss in Elasmobranchii (sharks and rays). Compared to other vertebrate groups, the chimaera PXR ortholog displays a diverse expression pattern (skin and gills) and a unique activation profile by classical xenobiotic ligands. Our findings provide insights into the molecular landscape of detoxification mechanisms and suggest lineage-specific adaptations in response to xenobiotics in gnathostome evolution.


Assuntos
Elasmobrânquios/classificação , Elasmobrânquios/genética , Evolução Molecular , Redes Reguladoras de Genes , Filogenia , Receptor de Pregnano X/genética , Animais , Células COS , Cercopithecus aethiops , Genes Reporter , Inativação Metabólica/genética , Luciferases/metabolismo , Receptor de Pregnano X/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Sintenia/genética , Ativação Transcricional/genética
15.
Methods Mol Biol ; 1966: 101-105, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041741

RESUMO

Immunofluorescent staining (IF) uses antigen-antibody complexes tagged with fluorochromes to observe the expression of proteins within a tissue sample. Multiple groups have described optimized methods to visualize several proteins simultaneously within the same tissue section using immunofluorescence in both mouse and human FFPE tissues. Our group routinely uses an optimized protocol described here to examine nuclear receptor expression in experimental samples from conditional knockout in vivo studies.


Assuntos
Imunofluorescência/métodos , Expressão Gênica , Inclusão em Parafina , Receptores Citoplasmáticos e Nucleares/análise , Animais , Formaldeído , Humanos , Camundongos , Receptores Citoplasmáticos e Nucleares/genética
16.
Methods Mol Biol ; 1966: 163-173, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31041746

RESUMO

Reporter assays are useful to study nuclear receptor activation and for example to evaluate the propensity of novel drug candidates to cause induction of drug-metabolizing cytochrome P450 enzymes. Here, we describe a protocol for a reverse transfection system to study the activation of human nuclear receptors constitutive androstane receptor and pregnane X receptor. The system provides long-term stability and uniformity of DNA-carrier complexes, thus avoiding the inherent variation in conventional transfection methods. Further, the system is easily adaptable for different studies. It offers reproducible and reliable results for early drug development and mechanistic studies related to nuclear receptor activation and resulting changes in gene expression.


Assuntos
Hepatócitos/metabolismo , Receptor de Pregnano X/genética , Receptores Citoplasmáticos e Nucleares/genética , Transfecção/métodos , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/metabolismo , Genes Reporter , Humanos , Receptor de Pregnano X/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo
17.
Cell Physiol Biochem ; 52(4): 802-821, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30946556

RESUMO

BACKGROUND/AIMS: The rapid nuclear translocation of signaling proteins upon stimulation is important for the regulation of de-novo gene expression. However, the molecular mechanisms of this translocation is not well understood, although some studies suggest that much of this translocation may be mediated by beta-like importins (Imps). Here we undertook to study the stimulated nuclear shuttling of JNK and p38 MAPKs. METHODS: For this purpose, we used coimmunoprecipitation, proximity ligation assay, gel filtration and immunostaining to examine the mechanism of nuclear translocation of these proteins. RESULTS: We found that JNK and p38 MAPKs translocate into the nucleus in a Ran dependent, but NLS- or NTS-independent manner, unrelated to their catalytic activity. We show that this translocation involves three ß-like Imps, 3, 7 and 9. Knockdown of these Imps inhibits the nuclear translocation of the MAPKs, and thereby, phosphorylation of their transcription factor targets. We further demonstrate that the translocation requires the stimulated formation of heterotrimers composed of Imp3/Imp7/MAPK or Imp3/Imp9/MAPK. JNK1/2 and p38α/ß bind to either Imp7 or Imp9 upon stimulated post-translational modifications of the two Imps, while Imp3 joins the complex after its stimulation-induced phosphorylation. Once formed, these heterotrimers move to the nuclear envelope where Imp3 remains, while Imp7 or Imp9 escort the MAPKs into the nucleus. CONCLUSION: These results suggest that ß-like Imps are central mediators of stimulated nuclear translocation of signaling proteins, providing a central level of regulation of the induction of cellular processes such as transcription upon stimulation.


Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Sequência de Aminoácidos , Anisomicina/farmacologia , Núcleo Celular/metabolismo , Células HeLa , Humanos , Células MCF-7 , Microscopia de Fluorescência , Fosforilação , Ligação Proteica , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genética , Alinhamento de Sequência
18.
Int J Mol Sci ; 20(8)2019 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-31013627

RESUMO

Overcoming P-glycoprotein (P-gp) efflux is a strategy to improve the absorption and pharmacokinetics of its substrate drugs. Berberine inhibits P-gp and thereby increases the bioavailability of the P-gp substrate digoxin in rodents. However, the effects of berberine on P-gp in chickens are still unclear. Here, we studied the role of berberine in modulating broilers P-gp expression and function through both in situ and in vitro models. In addition, molecular docking was applied to analyze the interactions of berberine with P-gp as well as with chicken xenobiotic receptor (CXR). The results showed that the mRNA expression levels of chicken P-gp and CXR decreased in the ileum following exposure to berberine. The absorption rate constant of rhodamine 123 increased after berberine treatment, as detected using an in situ single-pass intestinal perfusion model. Efflux ratios of P-gp substrates (tilmicosin, ciprofloxacin, clindamycin, ampicillin, and enrofloxacin) decreased and the apparent permeability coefficients increased after co-incubation with berberine in MDCK-chAbcb1 cell models. Bidirectional assay results showed that berberine could be transported by chicken P-gp with a transport ratio of 4.20, and this was attenuated by verapamil (an inhibitor of P-gp), which resulted in a ratio of 1.13. Molecular docking revealed that berberine could form favorable interactions with the binding pockets of both CXR and P-gp, with docking scores of -7.8 and -9.5 kcal/mol, respectively. These results indicate that berberine is a substrate of chicken P-gp and down-regulates P-gp expression in chicken tissues, thereby increasing the absorption of P-gp substrates. Our findings suggest that berberine increases the bioavailability of other drugs and that drug-drug interactions should be considered when it is co-administered with other P-gp substrates with narrow therapeutic windows.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Berberina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Berberina/química , Galinhas , Cães , Células Madin Darby de Rim Canino , Modelos Moleculares , Conformação Proteica , RNA Mensageiro/genética , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Relação Estrutura-Atividade
19.
Epilepsy Res ; 153: 49-58, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30986657

RESUMO

The Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels are highly expressed in the Central Nervous Systems, where they are responsible for the Ih current. Together with specific accessory proteins, these channels finely regulate neuronal excitability and discharge activity. In the last few years, a substantial body of evidence has been gathered showing that modifications of Ih can play an important role in the pathogenesis of epilepsy. However, the extent to which HCN dysfunction is spread among the epileptic population is still unknown. The aim of this work is to evaluate the impact of genetic mutations potentially affecting the HCN channels' activity, using a NGS approach. We screened a large cohort of patients with epilepsy of unknown etiology for mutations in HCN1, HCN2 and HCN4 and in genes coding for accessory proteins (MiRP1, Filamin A, Caveolin-3, TRIP8b, Tamalin, S-SCAM and Mint2). We confirmed the presence of specific mutations of HCN genes affecting channel function and predisposing to the development of the disease. We also found several previously unreported additional genetic variants, whose contribution to the phenotype remains to be clarified. According to these results and data from literature, alteration of HCN1 channel function seems to play a major role in epilepsy, but also dysfunctional HCN2 and HCN4 channels can predispose to the development of the disease. Our findings suggest that inclusion of the genetic screening of HCN channels in diagnostic procedures of epileptic patients should be recommended. This would help pave the way for a better understanding of the role played by Ih dysfunction in the pathogenesis of epilepsy.


Assuntos
Epilepsia/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Mutação/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Caderinas/genética , Proteínas de Transporte/genética , Caveolina 3/genética , Estudos de Coortes , Eletroencefalografia , Saúde da Família , Feminino , Filaminas/genética , Testes Genéticos , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Receptores Citoplasmáticos e Nucleares/genética
20.
Gene ; 705: 167-176, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31026569

RESUMO

Hemorrhoid is a common and recurrent proctological disease, which is often accompanied by angiogenesis and edema. MicroRNAs in the DLK1-DIO3 imprinted clusters are involved in the development and pathogenesis of mammalian hemorrhoids. Results of the present study indicated multiple, differential expression of DLK1-DIO3 imprinted cluster microRNA between hemorrhoid and normal tissues, where miR-412-5p expression in hemorrhoid tissue was significantly decreased. Fluorescein reporter assays showed that miR-412-5p silenced Xpo1 mRNA expression by targeting its 3'-UTR. Overexpression of miR-412-5p in human umbilical vein endothelial cells (HUVECs) indicated that proliferation, migration and formation of vascular structures in HUVECs were inhibited in vitro. In addition, overexpression of miR-412-5p significantly inhibited Xpo1 expression and promoted upregulation of the p53 protein and its retention in the nucleus. Simultaneously, expression of p66SHC and p16 proteins was activated. In summary, downregulation of endogenous miR-412-5p expression in hemorrhoid vascular endothelial cells leads to high expression of the target gene Xpo1 and translocation of the p53 protein out of the nucleus, rendering it unable to activate p66SHC and p16. This ultimately weakens regulation of the vascular endothelial cell cycle, thereby accelerating the division of hemorrhoid vascular endothelial cells, leading to angiogenesis.


Assuntos
Hemorroidas/genética , Carioferinas/genética , MicroRNAs/genética , Neovascularização Patológica/genética , Receptores Citoplasmáticos e Nucleares/genética , Regiões 3' não Traduzidas , Adulto , Movimento Celular , Núcleo Celular/metabolismo , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Regulação para Baixo , Feminino , Regulação da Expressão Gênica , Impressão Genômica , Hemorroidas/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/metabolismo , Transdução de Sinais , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo , Proteína Supressora de Tumor p53/metabolismo
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