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1.
Cell Physiol Biochem ; 53(2): 366-387, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31385665

RESUMO

BACKGROUND/AIMS: The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase (MAPK) family. Upon stimulation, these kinases translocate from the cytoplasm to the nucleus, where they induce physiological processes such as proliferation and differentiation. The mechanism of translocation of this kinase involves phosphorylation of two Ser residues within a nuclear translocation signal (NTS), which allows binding to importin7 and a subsequent penetration via nuclear pores. However, the regulation of this process and the protein kinases involved are not yet clear. METHODS: To answer this point we developed specific anti phospho-SPS antibody, used this and other antibodies in Western blots and crystalized the phospho-mimetic mutated ERK. RESULTS: Here we show that the phosphorylation of both Ser residues is mediated mainly by casein kinase 2 (CK2) and that active ERK may assist in the phosphorylation of the N-terminal Ser. We also demonstrate that the phosphorylation is dependent on the release of ERK from cytoplasmic anchoring proteins. Crystal structure of the phosphomimetic ERK revealed that the NTS phosphorylation creates an acidic patch in ERK. Our model is that in resting cells ERK is bound to cytoplasmic anchors, which prevent its NTS phosphorylation. Upon stimulation, phosphorylation of the ERK TEY domain releases ERK and allows phosphorylation of its NTS by CK2 and active ERK to generate a negatively charged patch in ERK, binding to importin 7 and nuclear translocation. CONCLUSION: These results provide an important role of CK2 in regulating nuclear ERK activities.


Assuntos
Núcleo Celular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transporte Ativo do Núcleo Celular , Caseína Quinase II/metabolismo , Linhagem Celular , Humanos , Carioferinas/metabolismo , Fosforilação , Ligação Proteica , Receptores Citoplasmáticos e Nucleares/metabolismo
2.
Cell Physiol Biochem ; 53(1): 186-199, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31278696

RESUMO

BACKGROUND/AIMS: Estrogen could play a key role in the mechanisms underlying sex-related disparity in the incidence of thrombotic events. We investigated whether estrogen receptors (ERs) were expressed in human red blood cells (RBCs), and if they affected cell signaling of erythrocyte constitutive isoform of endothelial NO-synthase (eNOS) and nitric oxide (NO) release. METHODS: RBCs from 29 non-smoker volunteers (15 males and 14 females) aged between 20 and 40 years were analyzed by cytometry and western blot. In particular, content and distribution of ER-α and ER-ß, tyrosine kinases and eNOS phosphorylation and NO release were analyzed. RESULTS: We demonstrated that: i) both ER-α and ER-ß were expressed by RBCs; ii) they were both functionally active; and iii) ERs distribution and function were different in males and females. In particular, ERs modulated eNOS phosphorylation and NO release in RBCs from both sexes, but they induced the phosphorylation of specific tyrosine residues of kinases linked to eNOS activation and NO release in the RBCs from females only. CONCLUSION: Collectively, these data suggest that ERs could play a critical role in RBC intracellular signaling. The possible implication of this signaling in sex-linked risk disparity in human cardiovascular diseases, e.g. in thrombotic events, may not be ruled out.


Assuntos
Receptores Estrogênicos/metabolismo , Transdução de Sinais , Adulto , Dronabinol/farmacologia , Eritrócitos/citologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/antagonistas & inibidores , Receptor beta de Estrogênio/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação/efeitos dos fármacos , Piperidinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirazóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Adulto Jovem
3.
Nat Commun ; 10(1): 2824, 2019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-31249305

RESUMO

The fibrogenic response in tissue-resident fibroblasts is determined by the balance between activation and repression signals from the tissue microenvironment. While the molecular pathways by which transforming growth factor-1 (TGF-ß1) activates pro-fibrogenic mechanisms have been extensively studied and are recognized critical during fibrosis development, the factors regulating TGF-ß1 signaling are poorly understood. Here we show that macrophage hypoxia signaling suppresses excessive fibrosis in a heart via oncostatin-m (OSM) secretion. During cardiac remodeling, Ly6Chi monocytes/macrophages accumulate in hypoxic areas through a hypoxia-inducible factor (HIF)-1α dependent manner and suppresses cardiac fibroblast activation. As an underlying molecular mechanism, we identify OSM, part of the interleukin 6 cytokine family, as a HIF-1α target gene, which directly inhibits the TGF-ß1 mediated activation of cardiac fibroblasts through extracellular signal-regulated kinase 1/2-dependent phosphorylation of the SMAD linker region. These results demonstrate that macrophage hypoxia signaling regulates fibroblast activation through OSM secretion in vivo.


Assuntos
Fibrose/metabolismo , Hipóxia/metabolismo , Macrófagos/metabolismo , Oncostatina M/metabolismo , Animais , Antígenos Ly/genética , Antígenos Ly/metabolismo , Feminino , Fibroblastos/metabolismo , Fibrose/genética , Fibrose/patologia , Hipóxia/genética , Hipóxia/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Oncostatina M/genética , Fosforilação , Transdução de Sinais , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
4.
Biochemistry (Mosc) ; 84(5): 529-539, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31234767

RESUMO

Hypoxia plays a critical role in progression of atherosclerosis. Local oxygen deficiency in a plaque creates a specific microenvironment that alters the transcriptome of resident cells, particularly of macrophages. Reverse cholesterol transport from plaque to liver is considered a main mechanism for regression of atherosclerosis. Ubiquitously expressed ATP-binding cassette transporter A1 (ABCA1) and liver- and small intestine-derived apolipoprotein A-1 (ApoA-1) are two main actors in this process. We recently reported endogenous apoA-1 expression in human macrophages. While ABCA1 and ApoA-1 have antiatherogenic properties, the role of complement factor C3 is controversial. Plasma C3 level positively correlates with the risk of cardiovascular diseases. On the other hand, C3 gene knockout in a murine atherosclerosis model increases both plaque size and triglycerides level in blood. In the present study, we show for the first time that a hypoxia-mimicking agent, CoCl2, induces the upregulation of the apoA-1 and C3 genes and the accumulation of intracellular and membrane protein ApoA-1 in THP-1 macrophages. The MEK1/2-Erk1/2 and MKK4/7-JNK1/2/3 cascades are involved in upregulation of ABCA1 and C3 via activation of transcription factor NF-κB, which interacts with the HIF-1α subunit of hypoxia-inducible factor 1 (HIF-1). The three major MAP-kinase cascades (Erk1/2, JNK1/2/3, and p38) and the NF-κB transcription factor are involved in the hypoxia-induced expression of the apoA-1 gene in THP-1 macrophages.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Apolipoproteína A-I/metabolismo , Hipóxia Celular , Complemento C3/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Apolipoproteína A-I/genética , Linhagem Celular Tumoral , Cobalto/farmacologia , Complemento C3/análise , Complemento C3/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Regulação para Cima/efeitos dos fármacos
5.
DNA Cell Biol ; 38(7): 660-669, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31120332

RESUMO

To perform global transcriptome profiling using RNA-seq in the peripheral blood of intracerebral hemorrhage (ICH) patients. In 11 patients with ICH, peripheral blood was collected within 24 h of symptom onset or last known well, and a second blood draw occurred 72 h (±6) after the first. RNA-seq identified differentially expressed genes (DEGs) between the first and second samples. Biological pathway enrichment analysis was performed with Ingenuity® Pathway Analysis (IPA). A total of 16,640 genes were identified and 218 were significant DEGs after ICH (false discovery rate <0.1). IPA identified 97 disease and functional categories that were significantly upregulated (z-score >2) post-ICH; 46 categories were specifically related to immune cell activation, 22 to general cellular activation processes, and 4 to other inflammation-related responses. In the canonical pathway and network analysis, inflammatory mediators of particular importance included interleukin-8, NF-κB, ERK1/2, and members of the integrin class. ICH induced peripheral blood gene expression at 72 to 96 h compared with 0 to 24 h from symptom onset. DEGs that were highly expressed included those related to inflammation and activation of the immune response. Further research is needed to determine whether these changes affect outcomes and may represent new therapeutic targets.


Assuntos
Hemorragia Cerebral/genética , Transcriptoma , Adulto , Idoso , Hemorragia Cerebral/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Inflamação/genética , Interleucina-8/genética , Interleucina-8/metabolismo , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo
6.
Life Sci ; 229: 173-179, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31103606

RESUMO

AIMS: The innate immune response induced by bacterial peptidoglycan peptides, such as γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP), is an important host defense system. However, little is known about the innate immune response in the lung alveolar region. In this study, we examined induction of the innate immune response by iE-DAP in human alveolar epithelial cell lines, NCI-H441 (H441) and A549. MAIN METHODS: Induction of the innate immune response was evaluated by measuring the mRNA expression of cytokines and their release into the culture medium. KEY FINDINGS: iE-DAP treatment increased the mRNA expression of interleukin (IL)-6 and IL-8, and increased release of these pro-inflammatory cytokines into the culture medium in H441 cells, but not in A549 cells. Lack of release of these cytokines in A549 cells may have been due to lack of peptide transporter 2 (PEPT2) function. Intracellular nucleotide-binding oligomerization domain 1 (NOD1) recognizes iE-DAP and activates downstream signaling pathways to initiate the immune response. Therefore, the role of mitogen-activated protein kinase (MAPK) signaling pathways was examined in H441 cells. As a result of inhibition studies, receptor-interacting serine/threonine-protein kinase 2 and MAPK signaling pathways, such as p38 MAPK and extracellular signal-regulated kinase, but not c-Jun N-terminal kinase, were determined to be involved in the innate immune response in H441 cells. In addition, the nuclear factor κB pathway also played a role in the innate immune response. SIGNIFICANCE: These findings indicated that the innate immune response induced by bacterial peptides could occur in a PEPT2- and NOD1-dependent manner in alveolar epithelial cells.


Assuntos
Células Epiteliais Alveolares/imunologia , Ácido Diaminopimélico/análogos & derivados , Imunidade Inata/imunologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Simportadores/metabolismo , Células A549 , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Citocinas/metabolismo , Ácido Diaminopimélico/farmacologia , Humanos , Imunidade Inata/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Transdução de Sinais , Simportadores/genética
7.
Int J Mol Sci ; 20(9)2019 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-31052520

RESUMO

Mathematical optimization framework allows the identification of certain nodes within a signaling network. In this work, we analyzed the complex extracellular-signal-regulated kinase 1 and 2 (ERK1/2) cascade in cardiomyocytes using the framework to find efficient adjustment screws for this cascade that is important for cardiomyocyte survival and maladaptive heart muscle growth. We modeled optimal pharmacological intervention points that are beneficial for the heart, but avoid the occurrence of a maladaptive ERK1/2 modification, the autophosphorylation of ERK at threonine 188 (ERK Thr 188 phosphorylation), which causes cardiac hypertrophy. For this purpose, a network of a cardiomyocyte that was fitted to experimental data was equipped with external stimuli that model the pharmacological intervention points. Specifically, two situations were considered. In the first one, the cardiomyocyte was driven to a desired expression level with different treatment strategies. These strategies were quantified with respect to beneficial effects and maleficent side effects and then which one is the best treatment strategy was evaluated. In the second situation, it was shown how to model constitutively activated pathways and how to identify drug targets to obtain a desired activity level that is associated with a healthy state and in contrast to the maleficent expression pattern caused by the constitutively activated pathway. An implementation of the algorithms used for the calculations is also presented in this paper, which simplifies the application of the presented framework for drug targeting, optimal drug combinations and the systematic and automatic search for pharmacological intervention points. The codes were designed such that they can be combined with any mathematical model given by ordinary differential equations.


Assuntos
Cardiomegalia/tratamento farmacológico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Algoritmos , Cardiomegalia/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Cardiovasculares , Terapia de Alvo Molecular/métodos , Miócitos Cardíacos/patologia , Fosforilação/efeitos dos fármacos
8.
Int J Mol Sci ; 20(10)2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091671

RESUMO

The human Na+/H+ exchanger isoform 1 (NHE1) is a plasma membrane transport protein that plays an important role in pH regulation in mammalian cells. Because of the generation of protons by intermediary metabolism as well as the negative membrane potential, protons accumulate within the cytosol. Extracellular signal-regulated kinase (ERK)-mediated regulation of NHE1 is important in several human pathologies including in the myocardium in heart disease, as well as in breast cancer as a trigger for growth and metastasis. NHE1 has a N-terminal, a 500 amino acid membrane domain, and a C-terminal 315 amino acid cytosolic domain. The C-terminal domain regulates the membrane domain and its effects on transport are modified by protein binding and phosphorylation. Here, we discuss the physiological regulation of NHE1 by ERK, with an emphasis on the critical effects on structure and function. ERK binds directly to the cytosolic domain at specific binding domains. ERK also phosphorylates NHE1 directly at multiple sites, which enhance NHE1 activity with subsequent downstream physiological effects. The NHE1 cytosolic regulatory tail possesses both ordered and disordered regions, and the disordered regions are stabilized by ERK-mediated phosphorylation at a phosphorylation motif. Overall, ERK pathway mediated phosphorylation modulates the NHE1 tail, and affects the activity, structure, and function of this membrane protein.


Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Animais , Humanos , Fosforilação , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Trocador 1 de Sódio-Hidrogênio/química , Trocador 1 de Sódio-Hidrogênio/genética
9.
Mol Med Rep ; 19(6): 5039-5045, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059063

RESUMO

Wound healing is a dynamic process that involves highly coordinated cellular events, including proliferation and migration. Oral gingival fibroblasts serve a central role in maintaining oral mucosa homeostasis, and their functions include the coordination of physiological tissue repair. Recently, surface pre­reacted glass­ionomer (S­PRG) fillers have been widely applied in the field of dental materials for the prevention of dental caries, due to an excellent ability to release fluoride (F). In addition to F, S­PRG fillers are known to release several types of ions, including aluminum (Al), boron (B), sodium (Na), silicon (Si) and strontium (Sr). However, the influence of these ions on gingival fibroblasts remains unknown. The aim of the present study was to examine the effect of various concentrations of an S­PRG filler eluate on the growth and migration of gingival fibroblasts. The human gingival fibroblast cell line HGF­1 was treated with various dilutions of an eluent solution of S­PRG, which contained 32.0 ppm Al, 1,488.6 ppm B, 505.0 ppm Na, 12.9 ppm Si, 156.5 ppm Sr and 136.5 ppm F. Treatment with eluate at a dilution of 1:10,000 was observed to significantly promote the migration of HGF­1 cells. In addition, the current study evaluated the mechanism underlying the mediated cell migration by the S­PRG solution and revealed that it activated the phosphorylation of extracellular signal­regulated kinase 1/2 (ERK1/2), but not of p38. Furthermore, treatment with a MEK inhibitor blocked the cell migration induced by the solution. Taken together, these results suggest that S­PRG fillers can stimulate HGF­1 cell migration via the ERK1/2 signaling pathway, indicating that a dental material containing this type of filler is useful for oral mucosa homeostasis and wound healing.


Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Alumínio/química , Boro/química , Linhagem Celular , Movimento Celular , Proliferação de Células/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/metabolismo , Gengiva/citologia , Humanos , Íons/química , Íons/farmacologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Silício/química , Sódio/química , Estrôncio/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
10.
J Photochem Photobiol B ; 196: 111511, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31129510

RESUMO

Prolonged exposure of the skin to ultraviolet radiation (UV) leads to its damage and loss of protective properties. This condition called photoaging of the skin is caused by a number of destructive factors, such as reactive oxygen species (ROS) and proteolytic enzymes that cause damage to the extracellular matrix, e.g. collagen fibers. Many cells of the immune system, including neutrophils, are involved in the photoaging process. The presence of neutrophils in the skin exposed to UV irradiation is known; however, the mechanism of neutrophil activity at these conditions remains unclear. In our study, we focused on the ability of neutrophils to release neutrophil extracellular traps (NETs) and the role of these structures in the photoaging process. NET release occurs in response to various stimuli; however, we hereby showed that the UVA and UVB radiation that reaches the Earth's surface could activate the mechanism of netosis. UV-induced netosis was much faster than that activated by chemical or biological factors; however, it also occurred due to the production of ROS, known signal mediators in netosis. In this work, we also identified the probable netosis signaling pathway involved in the neutrophil response to UV. The participation of NET components may explain the ongoing process of skin photoaging, but it is also important to indicate netosis as a potential target for skin protection therapy. Antioxidants tested in this work, such as N-acetylcysteine, ethamsylate, as well as vitamin B1 (thiamine), can successfully inhibit UV-induced netosis, and thus be used as protective components against the negative effects of solar radiation.


Assuntos
Armadilhas Extracelulares/metabolismo , Neutrófilos/efeitos da radiação , Raios Ultravioleta , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Células Cultivadas , Armadilhas Extracelulares/química , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , Fosforilação/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Envelhecimento da Pele/efeitos da radiação , Quinase Syk/metabolismo
11.
Cell Physiol Biochem ; 52(6): 1381-1397, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31075189

RESUMO

BACKGROUND/AIMS: Ouabain, a well-known plant-derived toxin, is also a hormone found in mammals at nanomolar levels that binds to a site located in the a-subunit of Na⁺,K⁺-ATPase. Our main goal was to understand the physiological roles of ouabain. Previously, we found that ouabain increases the degree of tight junction sealing, GAP junction-mediated communication and ciliogenesis. Considering our previous results, we investigated the effect of ouabain on adherens junctions. METHODS: We used immunofluorescence and immunoblot methods to measure the effect of 10 nM ouabain on the cellular and nuclear content of E-cadherin, ß-catenin and γ-catenin in cultured monolayers of Marin Darby canine renal cells (MDCK). We also studied the effect of ouabain on adherens junction biogenesis through sequential Ca²âº removal and replenishment. Then, we investigated whether c-Src and ERK1/2 kinases are involved in these responses. RESULTS: Ouabain enhanced the cellular content of the adherens junction proteins E-cadherin, ß-catenin and γ-catenin and displaced ß-catenin and γ-catenin from the plasma membrane into the nucleus. Ouabain also increased the expression levels of E-cadherin and ß-catenin in the plasma membrane after Ca²âº replenishment. These effects on adherens junctions were sensitive to PP2 and PD98059, suggesting that they depend on c-Src and ERK1/2 signaling. The translocation of ß-catenin and γ-catenin into the nucleus was specific because ouabain did not change the localization of the tight junction proteins ZO-1 and ZO-2. Moreover, in ouabain-resistant MDCK cells, which express a Na⁺,K⁺-ATPase α1-subunit with low affinity for ouabain, this hormone was unable to regulate adherens junctions, indicating that the ouabain receptor that regulates adherens junctions is Na⁺,K⁺-ATPase. CONCLUSION: Ouabain (10 nM) upregulated adherens junctions. This novel result supports the proposition that one of the physiological roles of this hormone is the modulation of cell contacts.


Assuntos
Junções Aderentes/efeitos dos fármacos , Ouabaína/farmacologia , Junções Aderentes/metabolismo , Animais , Caderinas/metabolismo , Cálcio/metabolismo , Núcleo Celular/metabolismo , Cães , Células Madin Darby de Rim Canino , Microscopia de Fluorescência , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , beta Catenina/metabolismo , gama Catenina/metabolismo , Quinases da Família src/metabolismo
12.
Cell Physiol Biochem ; 52(6): 1398-1411, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31075190

RESUMO

BACKGROUND/AIMS: Visfatin is known to act as a mediator in several metabolic disorders, such as obesity, diabetes, and cardiovascular diseases. This study aimed to investigate the effect of visfatin on the adhesion of THP-1 monocytes to human vascular endothelial cells and the underlying mechanism. METHODS: Monocytes adhesion to endothelial cells was determined by using fluorescence-labeled monocytes. ICAM-1 and VCAM-1 expression in endothelial cells were measured by western blotting. Production of reactive oxygen species (ROS) was measured by using a fluorescent dye. The amounts of nuclear factor-kappa B (NF-κB) and phosphorylation of inhibitory factor of NF-κB (IκB) were determined by using western blot analysis. The translocation of NF-κB from the cytoplasm to the nucleus was determined by using immunofluorescence. RESULTS: Here we showed that visfatin significantly caused the upregulation of intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells, as well as enhanced monocyte adhesion to endothelial cells. Moreover, we found that inhibition of PI3K, Akt, and p38 MAPK activation significantly prevented visfatin-enhanced expression of ICAM-1 and VCAM-1 and monocyte adhesion to endothelial cells. Visfatin enhanced ROS production and IKK/NF-кB activation and then led to upregulation of ICAM-1 and VCAM-1 and enhanced monocyte adhesion to endothelial cells. These effects were also p38/PI3K/Akt-dependent. CONCLUSION: These results demonstrated that visfatin promoted monocyte-endothelial cell adhesion by increasing ICAM-1 and VCAM-1 expression via the activation of p38/PI3K/Akt signaling and downstream ROS production and IKK/NF-кB activation.


Assuntos
Adesão Celular/efeitos dos fármacos , Nicotinamida Fosforribosiltransferase/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Monócitos/citologia , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
Life Sci ; 230: 89-96, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31129138

RESUMO

AIMS: Extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) is a key downstream gene of Ras pathway. Activation of Ras-ERK1/2 has been testified to be linked to the progression of diverse cancers. Nonetheless, whether Ras-ERK1/2-tumorigenic pathway is mediated by epigenetic factors remains indistinct. The purpose of the research attempted to disclose the functions of H2BK5ac in Ras-ERK1/2-evoked CRC cell phenotypes. MATERIALS AND METHODS: Western blot assay was implemented for exploration of the relevancy between Ras-ERK1/2 and H2BK5ac. H2BK5Q was established and its functions in cell viability, colony formation and migration were appraised via utilizing MTT, soft-agar colony formation and Transwell assays. The mRNA and transcription of ERK1/2 downstream genes were estimated via RT-qPCR and ChIP assays. HDAC2 functions in SW48 cell phenotypes were evaluated after co-transfection with pEGFP-RasQ61L/T35S and si-HDAC2 vectors. Additionally, the involvements of ATF2 and MDM2 in Ras-ERK1/2-affected H2BK5ac expression were estimated. KEY FINDINGS: H2BK5ac expression was evidently repressed by Ras-ERK1/2 pathway in SW48 cells. Moreover, Ras-ERK1/2-elevated cell viability, the number of colonies and migration were both impeded by H2BK5ac. The mRNA and transcriptions of CYR61, IGFBP3, WNT16B, NT5E, GDF15 and CARD16 were both mediated by H2BK5ac. Additionally, HDAC2 silence overtly recovered H2BK5ac expression inhibited by Ras-ERK1/2, meanwhile abated Ras-ERK1/2-affected SW48 cell phenotypes. Beyond that, restrained H2BK5ac induced by Ras-ERK1/2 was concerned with MDM2-mediated ATF2 degradation. SIGNIFICANCE: These investigations testified that Ras-ERK1/2 pathway affected SW48 cell phenotypes through repressing H2BK5ac expression. Otherwise, declined H2BK5ac might be linked to MDM2-mediated ATF2 degradation.


Assuntos
Neoplasias Colorretais/metabolismo , Histonas/metabolismo , Histonas/fisiologia , Acilação , Carcinogênese , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/genética , Sobrevivência Celular , Neoplasias Colorretais/fisiopatologia , Progressão da Doença , Fator 15 de Diferenciação de Crescimento/metabolismo , Histona Desacetilase 2/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais , Proteínas ras/fisiologia
14.
Chem Biol Interact ; 307: 105-115, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31054283

RESUMO

Neutral endopeptidase (NEP) is an enzyme implicated in development of different tumors, e.g. colorectal cancer (CRC). In this study, the anti-cancer effects of NEP inhibitors, thiorphan (synthetic compound) and sialorphin (naturally occurring pentapeptide) on CRC cells were investigated. Moreover, we synthesized some derivatives of sialorphin (alanine scan analogues: AHNPR, QANPR, QHAPR, QHNAR; N-acetylated sialorphin; C-amidated sialorphin, and C-amidated alanine scan analogues) to examine the biological activity of these inhibitors on CRC cells. The cytotoxic activity of the NEP inhibitors against CRC cell lines (SW620 and LS180) and normal human fibroblasts (HSF) was evaluated. Additionally, the influence of NEP inhibitors on proliferation, cell cycle progression, induction of apoptosis, and the level of phosphorylation of MAP kinases and mTORC1 signaling pathway proteins in CRC cells were examined. The NEP inhibitors were non-cytotoxic to HSF cells; however, most of them slightly decreased the viability and inhibited proliferation of CRC cells. The N-acetylation or C-amidation of sialorphin or its alanine scan analogues resulted in decreased or abolished anti-proliferative activity of the NEP inhibitors towards the CRC cells. Additionally, thiorphan and sialorphin enhanced the anti-proliferative activity of other CRC-cell growth inhibitors (atrial natriuretic peptide-ANP and melphalan-MEL). The mechanisms involved in the anti-proliferative effects of the tested inhibitors were mediated via NEP and associated with induction of cell cycle arrest in the G0/G1 phase, increased activity of ERK1/2, and a reduced level of phosphorylation of mTOR (Ser2448), 4E-BP1, and p70S6K. However, the NEP inhibitors did not induce apoptosis in the CRC cells. These results have indicated that thiorphan and sialorphin or its derivatives AHNPR, QANPR, QHAPR, and QHNAR have the potential to be used as agents in treatment of patients with CRC.


Assuntos
Proliferação de Células/efeitos dos fármacos , Endopeptidases/metabolismo , Peptídeos/farmacologia , Inibidores de Proteases/farmacologia , Tiorfano/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Endopeptidases/química , Endopeptidases/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Peptídeos/síntese química , Peptídeos/química , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteases/química , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Tiorfano/química
15.
Nat Commun ; 10(1): 2030, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31048689

RESUMO

Acquired resistance to MEK1/2 inhibitors (MEKi) arises through amplification of BRAFV600E or KRASG13D to reinstate ERK1/2 signalling. Here we show that BRAFV600E amplification and MEKi resistance are reversible following drug withdrawal. Cells with BRAFV600E amplification are addicted to MEKi to maintain a precise level of ERK1/2 signalling that is optimal for cell proliferation and survival, and tumour growth in vivo. Robust ERK1/2 activation following MEKi withdrawal drives a p57KIP2-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death, selecting against those cells with amplified BRAFV600E. p57KIP2 expression is required for loss of BRAFV600E amplification and reversal of MEKi resistance. Thus, BRAFV600E amplification confers a selective disadvantage during drug withdrawal, validating intermittent dosing to forestall resistance. In contrast, resistance driven by KRASG13D amplification is not reversible; rather ERK1/2 hyperactivation drives ZEB1-dependent epithelial-to-mesenchymal transition and chemoresistance, arguing strongly against the use of drug holidays in cases of KRASG13D amplification.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Benzimidazóis/farmacologia , Benzimidazóis/uso terapêutico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Amplificação de Genes/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 2/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias/genética , Inibidores de Proteínas Quinases/uso terapêutico , Suspensão de Tratamento , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo
16.
Chemosphere ; 227: 43-52, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30981969

RESUMO

Cyanobacterial toxin cylindrospermopsin (CYN) is an emerging freshwater contaminant, whose expanding environmental occurrence might result into increased human health risks. CYN is potent hepatotoxin, with cytotoxicity and genotoxicity documented in primary hepatocytes or hepatoma cell lines. However, there is only limited information about CYN effects on adult human liver stem cells (LSCs), which play an important role in liver tissue development, regeneration and repair. In our study with human liver cell line HL1-hT1 which expresses characteristics of LSCs, CYN was found to be cytotoxic and increasing cell death after 24-48 h exposure to concentrations >1 µM. Subcytotoxic 1 µM concentration did not induce cell death or membrane damage, but inhibited cellular processes related to energy production, leading to a growth stagnation after >72 h. Interestingly, these effects were not associated with increased DNA damage, reactive oxygen species production, or endoplasmic reticulum stress. However, CYN induced a sustained (24-48 h) activation of mitogen-activated protein kinases ERK1/2 and p38, and increased expression of stress-related transcription factor ATF3. Thus, LSCs were not primarily affected by CYN-induced genotoxicity and oxidative stress, but via activation of signaling and transcriptional pathways critical for regulation of cell proliferation, stress responses, cell survival and inflammation. Alterations of LSCs during CYN-induced liver injury, including the role of nongenotoxic mechanisms, should be therefore considered in mechanistic assessments of chronic CYN hepatotoxicity and hepatocarcinogenicity.


Assuntos
Toxinas Bacterianas/toxicidade , Uracila/análogos & derivados , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Linhagem Celular , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Hepatócitos/efeitos dos fármacos , Humanos , Fígado/metabolismo , Sistema de Sinalização das MAP Quinases , Toxinas Marinhas , Microcistinas , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Células-Tronco , Testes de Toxicidade , Uracila/toxicidade
17.
Gastroenterology ; 157(1): 179-192.e2, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30930024

RESUMO

BACKGROUND & AIMS: Reduced gastrointestinal (GI) motility is a feature of disorders associated with intestinal dysbiosis and loss of beneficial microbes. It is not clear how consumption of beneficial commensal microbes, marketed as probiotics, affects the enteric nervous system (ENS). We studied the effects of the widely used probiotic and the commensal Lactobacillus rhamnosus GG (LGG) on ENS and GI motility in mice. METHODS: Conventional and germ free C57B6 mice were gavaged with LGG and intestinal tissues were collected; changes in the enteric neuronal subtypes were assessed by real-time polymerase chain reaction, immunoblots, and immunostaining. Production of reactive oxygen species (ROS) in the jejunal myenteric plexi and phosphorylation (p) of mitogen-activated protein kinase 1 (MAPK1) in the enteric ganglia were assessed by immunoblots and immunostaining. Fluorescence in situ hybridization was performed on jejunal cryosections with probes to detect formyl peptide receptor 1 (FPR1). GI motility in conventional mice was assessed after daily gavage of LGG for 1 week. RESULTS: Feeding of LGG to mice stimulated myenteric production of ROS, increased levels of phosphorylated MAPK1, and increased expression of choline acetyl transferase by neurons (P < .001). These effects were not observed in mice given N-acetyl cysteine (a ROS inhibitor) or LGGΩSpaC (an adhesion-mutant strain of LGG) or FPR1-knockout mice. Gavage of mice with LGG for 1 week significantly increased stool frequency, reduced total GI transit time, and increased contractions of ileal circular muscle strips in ex vivo experiments (P < .05). CONCLUSIONS: Using mouse models, we found that LGG-mediated signaling in the ENS requires bacterial adhesion, redox mechanisms, and FPR1. This pathway might be activated to increase GI motility in patients.


Assuntos
Motilidade Gastrointestinal/fisiologia , Trânsito Gastrointestinal/fisiologia , Íleo/metabolismo , Jejuno/metabolismo , Lactobacillus rhamnosus , Plexo Mientérico/metabolismo , Neurônios/metabolismo , Probióticos , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Animais , Antioxidantes/farmacologia , Colina O-Acetiltransferase/metabolismo , Sistema Nervoso Entérico/citologia , Sistema Nervoso Entérico/metabolismo , Motilidade Gastrointestinal/efeitos dos fármacos , Trânsito Gastrointestinal/efeitos dos fármacos , Vida Livre de Germes , Íleo/efeitos dos fármacos , Íleo/inervação , Hibridização in Situ Fluorescente , Jejuno/efeitos dos fármacos , Jejuno/inervação , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Contração Muscular/efeitos dos fármacos , Plexo Mientérico/citologia , Neurônios/efeitos dos fármacos , Fosforilação , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Formil Peptídeo/genética
18.
Artif Cells Nanomed Biotechnol ; 47(1): 1207-1215, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30942624

RESUMO

Histone H4 acetylation at lysine 12 (H4K12ac) has been reported to be associated with the poor prognosis of pancreatic cancer. The study intends to study whether H4K12ac participates in regulating the carcinogenic effect of Ras-ERK1/2 on osteosarcoma (OS). The plasmids of pEGFP-N1, pEGFP-RasWT and pEGFP-K-RasG12V/T35S were transfected into MG-63 cells, the protein levels of H4K12ac and ERK1/2 were analyzed by Western blot. Effects of H4K12ac on cell proliferation and migration of MG-63 cells were tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide solution (MTT), transwell, soft-agar colony formation and flow cytometry assays. Effect of H4K12ac on the transcription of ERK1/2 downstream genes was analyzed by RT-qPCR and ChIP assays. The involvements of HDAT6, HAT1 and MDM2 in cell proliferation and migration of MG-63 cells were finally studied. We found that H4K12ac was specifically down-regulated by Ras-ERK1/2 activation in MG-63 cells. H4K12ac suppressed Ras-ERK1/2-induced cell viability, colony formation and migration in MG-63 cells. Additionally, HDAC6 silence recovered Ras-ERK1/2-repressed H4K12ac expression, as well as inhibited the carcinogenic effect of Ras-ERK1/2 on MG-63 cells. Besides, down-regulated H4K12ac induced by Ras-ERK1/2 was found to be associated with MDM2-mediated HAT1 degradation. In conclusion, these results testified that Ras-ERK1/2 signalling promoted the development of OS by mediating H4K12ac through MDM2-mediated HAT1 degradation.


Assuntos
Histona Acetiltransferases/metabolismo , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Sistema de Sinalização das MAP Quinases , Osteossarcoma/patologia , Acetilação , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Ativação Enzimática , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transcrição Genética , Proteínas ras/metabolismo
19.
Chem Biol Interact ; 306: 110-116, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30991045

RESUMO

MicroRNAs (miRNAs) have been implicated in both biological and pathological processes in patients with systemic lupus erythematosus (SLE). Previous studies have demonstrated dysregulated expression of miR-199-3p, interleukin (IL)-10, and poly (ADP-ribose) polymerase-1 (PARP-1) in SLE. However, the underlying mechanisms of these aberrations have not been fully elucidated. In this study, we investigated the mechanism through which miR-199-3p dysregulation contributed to the pathogenesis of SLE. Altered gene expression was assessed by ChIP analysis. We then silenced the expression of candidate genes using siRNA for functional analysis; mRNA expression, protein levels, and protein expression were determined by qRT-PCR, ELISA, and western blotting, respectively. According to ChIP and qRT-PCR results, miR-199-3p was up-regulated in SLE patients. Moreover, IL-10 was found to be highly expressed in SLE patients by ELISA. Further, PARP1 was significantly down-regulated in SLE patients based on western blotting. Our results also indicated that miR-199-3p inhibits PARP1 expression by activating the ERK1/2 pathway, thereby increasing IL-10 expression. Significantly up-regulated miR-199-3p was inversely related to PARP-1 expression and positively correlated with IL-10 levels in SLE. As miR-199-3p was shown to target PARP-1 to activate the ERK1/2 pathway and promote IL-10 production, restoring physiological miR-199-3p levels could represent a potential therapeutic strategy for SLE treatment.


Assuntos
Interleucina-10/biossíntese , Lúpus Eritematoso Sistêmico/metabolismo , MicroRNAs/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Adulto , Feminino , Células HEK293 , Humanos , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/genética , Masculino , Pessoa de Meia-Idade , Adulto Jovem
20.
Life Sci ; 225: 55-63, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30946839

RESUMO

BACKGROUND/AIMS: Acetylation of H4 at lysine 16 (H4K16ac) has been well-characterized as an acetylated mark, and the expression of which is closely associated with the tumorigenesis of human cancers. This study aimed to reveal whether KRas mutation drives the initiation and progression of glioma via modulation of H4 acetylation. METHODS: Changes of H4K16 acetylation in human glioblastoma A172 cells following transfection with a plasmid for expression of mutant KRas were tested by western blot analysis. MTT assay, transwell assay, soft-agar colony formation assay, RT-PCR and chromatin immunoprecipitation were carried out to evaluate the effect of H4K16ac on A172 cells growth and migration. Furthermore, the enzymes participating in the deacetylation of H4K16ac were studied by using RT-PCR and western blot analysis. RESULTS: H4K16ac was found to be deacetylated by KRas-ERK1/2 activation. H4K16Q (a plasmid for mimicking H4K16ac) repressed A172 cells viability, colony formation, and migratory capacity. Besides, H4K16ac was capable of regulating the transcription of several ERK1/2 pathway downstream genes. KRas-ERK1/2 signaling repressed H4 acetylation at K16 via modulation of a histone deacetylase Sirt2, as well as a histone acetyl-transferase TIP60. Moreover, KRas-ERK1/2 inhibited TIP60 via an MDM2-dependnet fashion. CONCLUSION: Our results suggest that activation of KRas-ERK1/2 signaling participates in the onset and progression of glioma at least partially through modulating acetylation of H4 at K16. KRas-ERK1/2 signaling mediates the acetylation of H4K16 via Sirt2 and MDM2-dependnet degeneration of TIP60.


Assuntos
Glioma/patologia , Histonas/metabolismo , Lisina/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Acetilação , Apoptose , Movimento Celular , Proliferação de Células , Glioma/genética , Glioma/metabolismo , Histonas/genética , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais , Células Tumorais Cultivadas
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