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1.
Cell Physiol Biochem ; 53(4): 687-700, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31577078

RESUMO

BACKGROUND/AIMS: Apelin and its G protein-coupled receptor APJ (gene symbol Aplnr) are strongly expressed in magnocellular vasopressinergic neurons suggesting that the apelin/APJ system plays a key role at the central level in regulating salt and water balance by counteracting the antiduretic action of vasopressin (AVP). Likewise, recent studies revealed that apelin exerts opposite effects to those of vasopressin induced on water reabsorption via a direct action on the kidney collecting duct. However, the underlying mechanisms of the peripheral action of apelin are not clearly understood. Here, we thus investigated the role of the apelin/APJ system in the regulation of water balance in the kidney, and more specifically its involvement in modulating the function of aquaporin-2 (AQP2) in the collecting duct. METHODS: Mouse cortical collecting duct cells (mpkCCD) were incubated in the presence of dDAVP and treated with or without apelin-13. Changes in AQP2 expression and localization were determined by immunoblotting and confocal immunofluorescence staining. RESULTS: Herein, we showed that the APJ was present in mpkCCD cells. Treatment of mpkCCD with apelin-13 reduced the cAMP production and antagonized the AVP-induced increase in AQP2 mRNA and protein expressions. Immunofluorescent experiments also revealed that the AVP-induced apical cell surface expression of AQP2, and notably its phosphorylated isoform AQP2-pS269, was considerably reduced following apelin-13 application to mpkCCD cells. CONCLUSION: Our data reinforce the aquaretic role of the apelin/APJ system in the fine regulation of body fluid homeostasis at the kidney level and its physiological opposite action to the antiduretic activity of AVP.


Assuntos
Aquaporina 2/metabolismo , Desamino Arginina Vasopressina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Transporte Proteico/efeitos dos fármacos , Animais , Receptores de Apelina/metabolismo , Aquaporina 2/genética , Linhagem Celular , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos
2.
Cell Physiol Biochem ; 53(2): 400-412, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31403270

RESUMO

BACKGROUND/AIMS: Mutations in ABCA4 cause Stargardt macular degeneration, which invariably ends in legal blindness. We studied two common mutants, A1038V (in NBD1) and G1961E (in NBD2), with the purpose of exploring how they interact with the cell's quality control mechanism. The study was designed to determine how these mutants can be rescued. METHODS: We expressed wt and mutant ABCA4 in HEK293 cells and studied the effect of the mutations on trafficking and processing and the ability of correctors to rescue them. We used a combination of western blotting, confocal microscopy and surface biotinylation coupled with pulldown of plasma membrane proteins. RESULTS: G1961E is sensitive to inhibitors of the aggresome, tubacin and the lysosome, bafilomycin A. Both mutants cause a reduction in heat shock protein, Hsp27. Incubation of HEK293 cells expressing the mutants with VX-809, an FDA approved drug for the treatment of cystic fibrosis, increased the levels of A1038V and G1961E by 2- to 3-fold. Importantly, VX-809 increased the levels of both mutants at the plasma membrane suggesting that trafficking had been restored. Transfecting additional Hsp27 to the cells also increased the steady state levels of both mutants. However, in combination with VX-809 the addition of Hsp27 caused a dramatic increase in the protein expression particularly in the G1961 mutant which increased approximately 5-fold. CONCLUSION: Our results provide a new mechanism for the rescue of ABCA4 trafficking mutants based on the restoration of Hsp27. Our results provide a pathway for the treatment of Stargardt disease.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Aminopiridinas/farmacologia , Benzodioxóis/farmacologia , Transportadores de Cassetes de Ligação de ATP/genética , Aminopiridinas/uso terapêutico , Anilidas/farmacologia , Benzodioxóis/uso terapêutico , Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Leupeptinas/farmacologia , Lisossomos/metabolismo , Degeneração Macular/congênito , Degeneração Macular/tratamento farmacológico , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Mutação , Transporte Proteico/efeitos dos fármacos
3.
Bioengineered ; 10(1): 282-291, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31311401

RESUMO

Transforming growth factor (TGF)-ß1 plays a crucial role in the epithelial-to-mesenchymal transition (EMT) in many cancer types and in thyroid cancers. Epigallocatechin-3-gallate (EGCG), the most important ingredient in the green tea, has been reported to possess antioxidant and anticancer activities. However, the cellular and molecular mechanisms explaining its action have not been completely understood. In this study, we found that EGCG significantly suppresses EMT, invasion and migration in anaplastic thyroid carcinoma (ATC) 8505C cells in vitro by regulating the TGF-ß/Smad signaling pathways. EGCG significantly inhibited TGF-ß1-induced expression of EMT markers (E-cadherin reduction and vimentin induction) in 8505C cells in vitro. Treatment with EGCG completely blocked the phosphorylation of Smad2/3, translocation of Smad4. Taken together, these results suggest that EGCG suppresses EMT and invasion and migration by blocking TGFß/Smad signaling pathways.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Catequina/análogos & derivados , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Transdução de Sinais/efeitos dos fármacos , Células Epiteliais da Tireoide/efeitos dos fármacos , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/antagonistas & inibidores , Caderinas/genética , Caderinas/metabolismo , Catequina/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Humanos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína Smad3/genética , Proteína Smad3/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Células Epiteliais da Tireoide/metabolismo , Células Epiteliais da Tireoide/patologia , Fator de Crescimento Transformador beta1/farmacologia , Vimentina/agonistas , Vimentina/genética , Vimentina/metabolismo
4.
Environ Pollut ; 252(Pt B): 1216-1224, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31252119

RESUMO

The effects of methylamine on human health have been debated for several years, but the exact adverse outcomes and definite signaling cascades have not been elucidated yet. Herein, a NF-κB signal pathway, a positive regulator of inflammation was identified as the main pathway of methylamine exposure induced adverse effects in bronchial airway cells (16HBE) for the first time. The results indicated that methylamine could stimulate the overproduction of reactive oxygen species (ROS) in cytoplasm and mitochondria of 16HBE cells. Moreover, ROS accelerate the translocation and phosphorylation of NF-κB in nucleic and promote the expression of inflammatory, such as IL-8 and IL-6. As a result, methylamine was found to be increased ROS-mediated NF-κB activation in cells, leading to the production of inflammatory cytokine. Furthermore, the results also showed that methylamine could affect the expression of cytokines related genes, p53, STAT3, Bcl2, c-myc, Cyclin D, Hes1, Mcl-1, TGF-ß2. The breakdown of those cell proliferation and apoptosis related genes were leading to a common toxic mechanism of cell death. In summary, our work uncovers a mechanism by which methylamine can induce the formation of inflammation response and demonstrates potential inflammation and carcinogenesis in human airway cell upon the methylamine inhaled.


Assuntos
Poluentes Atmosféricos/toxicidade , Exposição Ambiental/efeitos adversos , Inflamação/patologia , Pneumopatias/induzido quimicamente , Metilaminas/toxicidade , Fator de Transcrição RelA/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Regulação da Expressão Gênica , Humanos , Interleucina-6/biossíntese , Interleucina-8/biossíntese , Mitocôndrias/metabolismo , Fosforilação , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
5.
Food Chem Toxicol ; 131: 110537, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31150782

RESUMO

Programmed death ligand-1 (PD-L1) is an important immune checkpoint for cancer immunotherapy in clinic. In this study, we reported that platycodin D, a natural product isolated from an edible and medicinal plant Platycodon grandiflorus (Jacq.) A. DC., down-regulated the protein level of PD-L1 in lung cancer cells. Flow cytometry and immunofluorescence assay showed a weaker surface PD-L1 signal in NCI-H1975 cells after the incubation with platycodin D (10 µM) for 15 min compared to the control group. Jurkat T cells showed enhancive interleukin-2 secretion when co-cultured with platycodin D-treated NCI-H1975 cells, suggesting that platycodin D-induced PD-L1 reduction increases the activation of Jurkat T cells. An augmentation of PD-L1 protein was detected in the cell culture medium from platycodin D treatment group. Chlorpromazine (60 µM) almost abolished the platycodin D-mediated PD-L1 extracellular release and restored the membrane PD-L1. Finally, hemolysis assay exhibited that platycodin D-triggered PD-L1 extracellular release was independent of the hemolytic mechanism. Taken together, our study demonstrates that platycodin D reduces the protein level of PD-L1 in lung cancer cells via triggering its release into the cell culture medium, which sheds new light for the application of natural products in cancer immunotherapy.


Assuntos
Antígeno B7-H1/metabolismo , Saponinas/farmacologia , Triterpenos/farmacologia , Linhagem Celular Tumoral , Clorpromazina/farmacologia , Humanos , Interleucina-2/metabolismo , Células Jurkat , Transporte Proteico/efeitos dos fármacos
6.
Cell Physiol Biochem ; 52(6): 1427-1445, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31088037

RESUMO

BACKGROUND/AIMS: Hydrophobic bile salts, such as glycochenodeoxycholate (GCDC) can trigger hepatocyte apoptosis, which is prevented by tauroursodesoxycholate (TUDC), but the effects of GCDC and TUDC on sinusoidal bile salt uptake via the Na⁺-taurocholate transporting polypeptide (Ntcp) are unclear. METHODS: The effects of GCDC and TUDC on the plasma membrane localization of Ntcp were studied in perfused rat liver by means of immunofluorescence analysis and super-resolution microscopy. The underlying signaling events were investigated by Western blotting and inhibitor studies. RESULTS: GCDC (20 µmol/l) induced within 60 min a retrieval of Ntcp from the basolateral membrane into the cytosol, which was accompanied by an activating phosphorylation of the Src kinases Fyn and Yes. Both, Fyn activation and the GCDC-induced Ntcp retrieval from the plasma membrane were sensitive to the NADPH oxidase inhibitor apocynin, the antioxidant N-acetylcysteine and the Src family kinase inhibitors SU6656 and PP-2, whereas PP-2 did not inhibit GCDC-induced Yes activation. Internalization of Ntcp by GCDC was also prevented by the protein kinase C (PKC) inhibitor Gö6850. TUDC (20 µmol/l) reversed the GCDC-induced retrieval of Ntcp from the plasma membrane and prevented the activation of Fyn and Yes in GCDC-perfused rat livers. Reinsertion of Ntcp into the basolateral membrane in GCDC-perfused livers by TUDC was sensitive to the protein kinase A (PKA) inhibitor H89 and the integrin-inhibitory peptide GRGDSP, whereas the control peptide GRADSP was ineffective. Ex posure of cultured rat hepatocytes to GCDC (50 µmol/l, 15min) increased the fluorescence intensity of the reactive oxygen fluorescent indicator DCF to about 1.6-fold of untreated controls in a TUDC (50 µmol/l)-sensitive way. GCDC caused a TUDC-sensitive canalicular dilatation without evidence for Bsep retrieval from the canalicular membrane. CONCLUSION: The present study suggests that GCDC triggers the retrieval of Ntcp from the basolateral membrane into the cytosol through an oxidative stress-dependent activation of Fyn. TUDC prevents the GCDC-induced Fyn activation and Ntcp retrieval through integrin-dependent activation of PKA.


Assuntos
Membrana Celular/metabolismo , Ácido Glicoquenodesoxicólico , Fígado/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Simportadores/metabolismo , Ácido Taurocólico , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Ácido Glicoquenodesoxicólico/metabolismo , Ácido Glicoquenodesoxicólico/farmacologia , Masculino , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Ácido Taurocólico/metabolismo , Ácido Taurocólico/farmacologia
7.
Int J Mol Sci ; 20(9)2019 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-31083521

RESUMO

ADP-ribosylation factor-guanine nucleotide exchange factors (ARF-GEFs) act as key regulators of vesicle trafficking in all eukaryotes. In Arabidopsis, there are eight ARF-GEFs, including three members of the GBF1 subfamily and five members of the BIG subfamily. These ARF-GEFs have different subcellular localizations and regulate different trafficking pathways. Until now, the roles of these BIG-subfamily ARF-GEFs have not been fully revealed. Here, analysis of the BIGs expression patterns showed that BIG3 and BIG5 have similar expression patterns. big5-1 displayed a dwarf growth and big3-1 big5-1 double mutant showed more severe defects, indicating functional redundancy between BIG3 and BIG5. Moreover, both big5-1 and big3-1 big5-1 exhibited a reduced sensitivity to Brassinosteroid (BR) treatment. Brefeldin A (BFA)-induced BR receptor Brassinosteroid insensitive 1 (BRI1) aggregation was reduced in big5-1 mutant, indicating that the action of BIG5 is required for BRI1 recycling. Furthermore, BR-induced dephosphorylation of transcription factor BZR1 was decreased in big3-1 big5-1 double mutants. The introduction of the gain-of-function of BZR1 mutant BZR1-1D in big3-1 big5-1 mutants can partially rescue the big3-1 big5-1 growth defects. Our findings revealed that BIG5 functions redundantly with BIG3 in plant growth and gravitropism, and BIG5 participates in BR signal transduction pathway through regulating BRI1 trafficking.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Gravitropismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Desenvolvimento Vegetal , Proteínas Quinases/metabolismo , Proteínas de Arabidopsis/genética , Brassinosteroides/farmacologia , Teste de Complementação Genética , Gravitropismo/efeitos dos fármacos , Proteínas de Fluorescência Verde/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Inflorescência/efeitos dos fármacos , Inflorescência/crescimento & desenvolvimento , Mutação/genética , Fenótipo , Desenvolvimento Vegetal/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
8.
J Headache Pain ; 20(1): 44, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31039731

RESUMO

BACKGROUND: Monoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor are efficacious for the prevention of migraine headaches. The downstream molecular mechanisms following ligand-receptor blockade by which these antibodies prevent CGRP signaling through CGRP receptors have not been demonstrated. METHODS: Here we produced tool monoclonal functional antagonist antibodies against CGRP and its canonical receptor and developed a novel cellular model using fluorogen-activated protein technology that allows detection of CGRP receptor internalization by flow cytometry and, for an extended time course, visualization by confocal microscopy. RESULTS: Using this cell model we showed that these antagonist antibodies block both CGRP-induced cAMP signaling and CGRP receptor internalization. At least 10-fold higher concentrations of either antibody are necessary to block CGRP receptor internalization compared with cAMP accumulation in our cell model. CONCLUSION: These data reinforce our understanding of how monoclonal functional antagonist antibodies interfere with CGRP signaling.


Assuntos
Anticorpos Monoclonais/metabolismo , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Células CHO , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina/farmacologia , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos Transgênicos , Transtornos de Enxaqueca/metabolismo , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
9.
Int J Mol Sci ; 20(9)2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31083359

RESUMO

The overactivation of microglia is known to trigger inflammatory reactions in the central nervous system, which ultimately induce neuroinflammatory disorders including Alzheimer's disease. However, increasing evidence has shown that menaquinone-4 (MK-4), a subtype of vitamin K2, can attenuate inflammation in the peripheral system. Whereas it was also observed at high levels within the brain, its function in this organ has not been well characterized. Therefore, we investigated the effect of MK-4 on microglial activation and clarified the underlying mechanism. Mouse microglia-derived MG6 cells were exposed to lipopolysaccharide (LPS) either with or without MK-4 pretreatment. Cell responses with respect to inflammatory cytokines (Il-1ß, Tnf-α, and Il-6) were measured by qRT-PCR. We further analyzed the phosphorylation of TAK1, IKKα/ß, and p65 of the NF-κB subunit by Western blotting. We observed that in LPS-induced MG6 cells, MK-4 dose-dependently suppressed the upregulation of inflammatory cytokines at the mRNA level. It also significantly decreased the phosphorylation of p65, but did not affect that TAK1 and IKKα/ß. Furthermore, the nuclear translocation of NF-κB in LPS-induced MG6 cells was inhibited by MK-4. These results indicate that MK-4 attenuates microglial inflammation by inhibiting NF-κB signaling.


Assuntos
Inflamação/metabolismo , Inflamação/patologia , Microglia/metabolismo , Microglia/patologia , NF-kappa B/metabolismo , Transdução de Sinais , Vitamina K 2/análogos & derivados , Animais , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Humanos , Inflamação/induzido quimicamente , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos , Camundongos , Microglia/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Vitamina K/análogos & derivados , Vitamina K 2/farmacologia
10.
Cell Prolif ; 52(4): e12639, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31127673

RESUMO

OBJECTIVES: Accumulating data show that gangliosides are involved in regulation of cell proliferation. Specific changes in gangliosides expression associated with growth density of cells have been documented in several cell lines. However, the function and the potential mechanism of ganglioside GM1 in contact inhibition of growth are not clear. MATERIALS AND METHODS: EdU incorporation assay and western blot were applied to detect the contact inhibition of growth in human mammary epithelial cells. GM1 manipulation of cell proliferation and epidermal growth factor receptor (EGFR) activation was investigated by immunoprecipitation, OptiPrep density gradient centrifugation and immunofluorescence. The function of GM1 on contact inhibition of growth was further studied by using GM1 stably knockdown and overexpression cells. RESULTS: MCF-10A, MCF-7 and MDA-MB-231 cells showed contact inhibition of growth in high-density condition. Exogenous addition of GM1 to high-density cells clearly inhibited cell growth and deactivated EGFR signalling. Compared to normal-density cells, distribution of EGFR in high-density cells was decreased in glycosphingolipid-enriched microdomain (GEM), but more concentrated in caveolae, and incubation with GM1 obviously promoted this translocation. Furthermore, the cell growth and EGFR activation were increased in GM1 stably knockdown cells and decreased in GM1 stably overexpression cells when cultured in high density. CONCLUSIONS: Our results demonstrated that GM1 suppressed EGFR signalling and promoted contact inhibition of growth by changing the localization of EGFR from GEM to caveolae.


Assuntos
Cavéolas/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Gangliosídeo G(M1)/farmacologia , Cavéolas/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Glicoesfingolipídeos/metabolismo , Humanos , Células MCF-7 , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
11.
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
12.
Pharm Biol ; 57(1): 245-249, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30929547

RESUMO

CONTEXT: Allicin is a potential antiarrhythmic agent. The antiarrhythmic properties of allicin rely on its blockade of various cardiac ion channels. The l-type calcium (Cav1.2) channel provides a pivotal substrate for cardiac electrophysiologic activities. The mechanism of allicin on Cav1.2 remains unclear. OBJECTIVE: This study evaluated the potential of allicin on the synthesis and trafficking of Cav1.2 channels. MATERIALS AND METHODS: Primary cardiomyocytes (CMs) from neonatal Sprague-Dawley (SD) rats were exposed to allicin (0, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100 µg/mL) for 24 and 48 h. The CellTiter-Glo assay was performed to measure CM viability. Western blot with grayscale analysis and confocal laser scanning microscopy were used to evaluate the effects of allicin on the synthesis and trafficking of Cav1.2 channel proteins in primary CMs. RESULTS: There was no significant difference in apoptotic toxicity from the actual cell viability (p > 0.05) in any group (0, 0.0001, 0.001, 0.01, 0.1, 1, 10, 100 µg/mL allicin), except that viability in the 0.001 and 0.01 µg/mL groups at 24 h were significantly greater (137.37 and 135.96%) (p < 0.05). Western blot with grayscale analysis revealed no substantial inhibition by allicin of the synthesis of Cav1.2 proteins. Confocal laser scanning microscopy revealed trafficking dysfunction of Cav1.2 channels caused by allicin in primary CMs. CONCLUSION: This study is the first to demonstrate that allicin inhibits cardiac Cav1.2 channels by disrupting trafficking, possibly mediating its antiarrhythmic benefits. Therefore, allicin might serve as a new antiarrhythmic agent in the future.


Assuntos
Antiarrítmicos/farmacologia , Canais de Cálcio Tipo L/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Ácidos Sulfínicos/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Masculino , Miócitos Cardíacos/metabolismo , Cultura Primária de Células , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
13.
Plant Mol Biol ; 100(1-2): 95-110, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31011887

RESUMO

KEY MESSAGE: Overexpression of VaWRKY12, whose nuclear translocation increased under low temperature, enhanced the cold tolerance of Arabidopsis and grapevine calli and significantly increased the expression of antioxidant-related genes. Low temperature causes injuries to buds during winter and to young shoots during early spring, thereby affecting grapevine quality and yield. Understanding the regulatory mechanisms of cold stress responses is essential for the breeding of new grapevine cultivars with excellent cold tolerance. Previous studies indicated that WRKY family genes are induced by low temperature in grapevine, but their function in cold stress responses was not clear. Here, a cold-induced WRKY gene, named VaWRKY12, was cloned from Vitis amurensis, which displays remarkable cold tolerance. An atypical transmembrane (TM) region was found in its C-terminal region. Transient expression assays showed that VaWRKY12 was localized in the nucleus and cytoplasm at normal temperature but only in the nucleus after cold treatment. By contrast, a truncated version of VaWRKY12 without the TM region was found specifically in the nucleus at normal temperature, and its binding activity to tandem W-box elements in yeast was stronger than that of VaWRKY12, indicating that the TM region might affect the location and function of VaWRKY12. Overexpression of VaWRKY12 enhanced the cold tolerance of transformed Arabidopsis and grapevine calli. Transcriptome data revealed that the expression of genes encoding antioxidant enzymes, including peroxidases and glutathione S-transferases, was upregulated after cold treatment in VaWRKY12-overexpressing grapevine calli compared to the control calli. This study identifies candidate target genes as a basis for further studies on the roles of VaWRKY12 in cold stress responses in grapevine.


Assuntos
Núcleo Celular/metabolismo , Temperatura Baixa , Proteínas de Plantas/metabolismo , Termotolerância/fisiologia , Fatores de Transcrição/metabolismo , Vitis/fisiologia , Sequência de Aminoácidos , Arabidopsis/genética , Núcleo Celular/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas , Filogenia , Reguladores de Crescimento de Planta/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Transporte Proteico/efeitos dos fármacos , Estresse Fisiológico/genética , Superóxido Dismutase/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Vitis/genética
14.
Int J Mol Sci ; 20(5)2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30866458

RESUMO

H. pylori is classified as a group I carcinogen by WHO because of its involvement in gastric cancer development. Several reports have suggested anti-bacterial effects of menadione, although the effect of menadione on major virulence factors of H. pylori and H. pylori-induced inflammation is yet to be elucidated. In this study, therefore, we demonstrated that menadione has anti-H. pylori and anti-inflammatory effects. Menadione inhibited growth of H. pylori reference strains and clinical isolates. Menadione reduced expression of vacA in H. pylori, and translocation of VacA protein into AGS (gastric adenocarcinoma cell) was also decreased by menadione treatment. This result was concordant with decreased apoptosis in AGS cells infected with H. pylori. Moreover, cytotoxin-associated protein A (CagA) translocation into H. pylori-infected AGS cells was also decreased by menadione. Menadione inhibited expression of several type IV secretion system (T4SS) components, including virB2, virB7, virB8, and virB10, that are responsible for translocation of CagA into host cells. In particular, menadione inhibited nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) activation and thereby reduced expression of the proinflammatory cytokines such as IL-1ß, IL-6, IL-8, and TNF-α in AGS as well as in THP-1 (monocytic leukemia cell) cell lines. Collectively, these results suggest the anti-bacterial and anti-inflammatory effects of menadione against H. pylori.


Assuntos
Infecções por Helicobacter/imunologia , Helicobacter pylori/efeitos dos fármacos , NF-kappa B/metabolismo , Vitamina K 3/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Calgranulina A/genética , Calgranulina A/metabolismo , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Infecções por Helicobacter/tratamento farmacológico , Infecções por Helicobacter/microbiologia , Helicobacter pylori/crescimento & desenvolvimento , Humanos , Testes de Sensibilidade Microbiana , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
15.
Int J Mol Sci ; 20(6)2019 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-30889901

RESUMO

The monocarbonyl analogue of curcumin (1E,4E)-1,5-Bis(2-methoxyphenyl)penta-1,4-dien-3-one (C1) has been used as a specific activator of the master gene transcription factor EB (TFEB) to correlate the activation of this nuclear factor with the increased activity of lysosomal glycohydrolases and their recruitment to the cell surface. The presence of active lysosomal glycohydrolases associated with the lipid microdomains has been extensively demonstrated, and their role in glycosphingolipid (GSL) remodeling in both physiological and pathological conditions, such as neurodegenerative disorders, has been suggested. Here, we demonstrate that Jurkat cell stimulation elicits TFEB nuclear translocation and an increase of both the expression of hexosaminidase subunit beta (HEXB), hexosaminidase subunit alpha (HEXA), and galactosidase beta 1 (GLB1) genes, and the recruitment of ß-hexosaminidase (Hex, EC 3.2.1.52) and ß-galactosidase (Gal, EC 3.2.1.23) on lipid microdomains. Treatment of Jurkat cells with the curcumin analogue C1 also resulted in an increase of both lysosomal glycohydrolase activity and their targeting to the cell surface. Similar effects of C1 on lysosomal glycohydrolase expression and their recruitment to lipid microdomains was observed by treating the SH-SY5Y neuroblastoma cell line; the effects of C1 treatment were abolished by TFEB silencing. Together, these results clearly demonstrate the existence of a direct link between TFEB nuclear translocation and the transport of Hex and Gal from lysosomes to the plasma membrane.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Membrana Celular/metabolismo , Curcumina/análogos & derivados , Curcumina/farmacologia , Hexosaminidases/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , beta-Galactosidase/metabolismo , Membrana Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Exocitose/efeitos dos fármacos , Humanos , Células Jurkat , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Fito-Hemaglutininas/farmacologia , Transporte Proteico/efeitos dos fármacos
16.
Phytomedicine ; 58: 152880, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30901661

RESUMO

BACKGROUND: Sijunzi decoction, a representative Chinese herbal formula of reinforcing Qi strengthening spleen, has been widely used for treating patients with gastrointestinal diseases and spleen-deficiency syndrome, however, the exact effects and mechanisms are remained unclear. HYPOTHESIS/PURPOSE: The migration of intestinal epithelial (IEC-6) cells has been demonstrated to be one of the major repair modalities during the healing of mucosal wounds. Ca2+ is the key factor in regulating cell migration. Thus, this study aimed to elucidate the potential effects and mechanisms of polysaccharides (SJZDP) extracted from Sijunzi decoction in intestinal mucosal restitution. METHOD: Cell migration was detected by scratch method with micropipette tip. Western blotting was adopted to evaluate the expression of STIM1 and STIM2 proteins. Immunofluorescence was carried out to assess the translocation of STIM1 protein. Immunoprecipitation was used to determine the levels of STIM1/TRPC1 and STIM1/STIM2 complexes. A indomethacin-induced intestinal mucosal injury rat model was applied. The content of polyamines in intestinal mucosa was detected by high-performance liquid chromatography. The morphological changes in the intestinal mucosa were observed at the end of animal experiment. RESULTS: The results showed that treatment with SJZDP significantly promoted cell migration, enhanced the level of STIM1 protein and STIM1/TRPC1 complexes, reduced the level of STIM2 protein and STIM1/STIM2 complexes. Further more, SJZDP exposure promoted the translocation of STIM1 to the plasma membrane. In vivo experiment results, the administration of SJZDP effectively reduced intestinal mucosal injury. CONCLUSION: These results revealed that SJZDP promotes intestinal epithelial restitution after wounding, presumably by regulating cellular levels of STIM1 and STIM2 differentially, stimulating the translocation of STIM1, and inducing STIM1/TRPC1 association as well as decreasing the level of STIM1/STIM2.


Assuntos
Cálcio/metabolismo , Medicamentos de Ervas Chinesas/química , Mucosa Intestinal/efeitos dos fármacos , Polissacarídeos/farmacologia , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Poliaminas/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos Sprague-Dawley , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/metabolismo
17.
Cell Struct Funct ; 44(1): 61-74, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30905923

RESUMO

Endocytosis mediates the internalization and ingestion of a variety of endogenous or exogenous substances, including virus particles, under the control of intracellular signaling pathways. We have previously reported that the complex formed between the small GTPase Ras and phosphoinositide 3-kinase (PI3K) translocates from the plasma membrane to endosomes, signaling from which thereby regulates clathrin-independent endocytosis, endosome maturation, influenza virus internalization, and infection. However, the molecular mechanism by which the Ras-PI3K complex is recruited to endosomes remains unclear. Here, we have identified the amino acid sequence responsible for endosomal localization of the Ras-PI3K complex. PI3K lacking this sequence failed to translocate to endosomes, and expression of the peptide comprising this PI3K-derived sequence inhibited clathrin-independent endocytosis, influenza virus internalization, and infection. Moreover, treatment of cells with this peptide in an arginine-rich, cell-penetrating form successfully suppressed influenza virus infection in vitro and ex vivo, making this peptide a potential therapeutic agent against influenza virus infection.Key words: signal transduction, endocytosis, endosome, imaging, influenza virus.


Assuntos
Endocitose/efeitos dos fármacos , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/fisiologia , Fragmentos de Peptídeos/farmacologia , Fosfatidilinositol 3-Quinase/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Humanos , Fragmentos de Peptídeos/química , Transporte Proteico/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Proteínas ras/metabolismo
18.
Cell Physiol Biochem ; 52(1): 76-93, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30790506

RESUMO

BACKGROUND/AIMS: Protein kinase C (PKC)- and RhoA/Rho-associated kinase (ROCK) play important roles in arterial sustained contraction. Although depolarization-elicited RhoA/ROCK activation is accepted, the role of PKC in depolarized vascular smooth muscle cells (VSMCs) is a subject of controversy. Our aim was to study the role of PKC in arterial contraction and its interaction with RhoA/ROCK. METHODS: Mass spectrometry was used to identify the PKC isoenzymes. PKCα levels and RhoA activity were analyzed by western blot and G-LISA, respectively, and isometric force was measured in arterial rings. RESULTS: In depolarized VSMCs RhoA and PKCα were translocated to the plasma membrane, where they colocalize and coimmunoprecipitate. Interestingly, depolarization-induced RhoA activation was downregulated by PKCα, effect reverted by PKCα inhibition. Phorbol 12,13-dibutyrate (PDBu) induced the translocation of PKCα to the plasma membrane, increased the level of RhoA in the cytosol and reduced RhoA/ROCK activity. These effects were reverted when PKC was inhibited. Pharmacological or siRNA inhibition of PKCα synergistically potentiated the vasorelaxant effect of RhoA/ROCK inhibition. CONCLUSION: The present study provides the first evidence that RhoA activity is downregulated by PKCα in depolarized and PDBu treated freshly isolated VSMCs and arteries, with an important physiological role on arterial contractility.


Assuntos
Membrana Celular/enzimologia , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Proteína Quinase C-alfa/metabolismo , Vasodilatação , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Masculino , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Dibutirato de 12,13-Forbol/farmacologia , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Wistar , Quinases Associadas a rho/metabolismo
19.
Toxicol Lett ; 306: 25-34, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30742880

RESUMO

Autophagy, which works to remove stress and maintain cellular homeostasis, is usually considered a "pro-survival" signal. Contrarily, apoptosis is programmed "pro-death" machinery. Polychlorinated biphenyls (PCBs) are a group of ubiquitous industrial pollutants. Our previous studies illustrated that a PCB quinone metabolite, PCB29-pQ, elicited both autophagy and apoptosis. However, the signaling underlying the autophagy and apoptosis cross-talk has not been characterized. Here, we found that PCB29-pQ-induced autophagy mainly occurred at a lower concentration (5 µM), while apoptosis mostly arose at a higher concentration (15 µM) in HepG2 cells. Next, we demonstrated the elevation of intracellular calcium levels and calpain activity with PCB29-pQ treatment; however, the unaffected subcellular location of truncated ATG5 and Beclin1 suggested the irrelevance of calpain towards the autophagy-to-apoptosis signaling shift. HMGB1 and p53 both serve as transcription factors that play crucial roles in the regulation of PCB29-pQ-induced autophagy and apoptosis. PCB29-pQ not only enhanced the expression of HMGB1 and p53 but also promoted their binding and cytosolic translocation. Interestingly, HMGB1 rather than p53 plays a primary role in 5 µM of PCB29-pQ-induced autophagy in the nucleus; however, p53 promoted apoptosis to a great extent in the cytosol at the dose of 15 µM PCB29-pQ. Together, HMGB1 and p53 provided a subtle balance between autophagy and apoptosis, thus determining the fate of PCB29-pQ-treated cells.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Poluentes Ambientais/toxicidade , Proteína HMGB1/metabolismo , Bifenilos Policlorados/toxicidade , Quinonas/toxicidade , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Cálcio/metabolismo , Calpaína/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Hep G2 , Humanos , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia
20.
Int J Mol Sci ; 20(4)2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30781399

RESUMO

Radiotherapy for treatment of hepatocellular carcinoma causes severe side effects, including acute hepatitis and chronic fibrosis. Complementary and alternative medicine (CAM) has emerged as an important part of integrative medicine in the management of diseases. Antrodia cinnamomea (AC), a valuable medicinal fungus originally found only in Taiwan, has been shown to possess anti-oxidation, vaso-relaxtation, anti-inflammation, anti-hepatitis, and anti-cancer effects. In this paper we evaluate the protective effects of ethanol extract of Antrodia cinnamomea (ACE) against radiotoxicity both in normal liver cell line CL48 and in tumor-bearing mice. In CL48, ACE protects cells by eliminating irradiation-induced reactive oxygen species (ROS) through the induction of Nrf2 and the downstream redox system enzymes. The protective effect of ACE was also demonstrated in tumor-bearing mice by alleviating irradiation-induced acute hepatitis. ACE could also protect mice from CCl4-induced hepatitis. Since both radiation and CCl4 cause free radicals, these results indicate that ACE likely contains active components that protect normal liver cells from free radical attack and can potentially benefit hepatocellular carcinoma (HCC) patients during radiotherapy.


Assuntos
Antrodia/química , Hepatite/tratamento farmacológico , Protetores contra Radiação/uso terapêutico , Doença Aguda , Animais , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoproteção/efeitos dos fármacos , Feminino , Depuradores de Radicais Livres/farmacologia , Hepatite/patologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/efeitos da radiação , Humanos , Inflamação/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Fator 2 Relacionado a NF-E2/metabolismo , Oxirredução , Transporte Proteico/efeitos dos fármacos , Protetores contra Radiação/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Soluções , Raios X
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