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Catecholamine stimulation over adrenergic receptors results in a state of hypercoagulability. Chronic stress involves the release and increase in circulation of catecholamines and other stress related hormones. Numerous observational studies in human have related stressful scenarios to several coagulation variables, but controlled stimulation with agonists or antagonists to adrenergic receptors are scarce. This systematic review is aimed at presenting an updated appraisal of the effect of adrenergic receptor modulation on variables related to human hemostasis by systematically reviewing the effect of adrenergic receptor-targeting drugs on scale variables related to hemostasis. By searching 3 databases for articles published between January 1st 2011 and February 16th, 2022 reporting effects on coagulation parameters from stimulation with α- or ß-adrenergic receptor targeting drugs in humans regardless of baseline condition, excluding records different from original research and those not addressing the main aim of this systematic review. Risk of bias assessed using the Revised Cochrane risk-of-bias tool for randomized trials (RoB 2). Tables describing a pro-thrombotic anti-fibrinolytic state induced after ß-adrenergic receptor agonist stimulation and the opposite after α1-, ß-adrenergic receptor antagonist stimulation were synthesized from 4 eligible records by comparing hemostasis-related variables to their baseline. Notwithstanding this low number of records, experimental interventions included were sound and mostly unbiased, results were coherent, and outcomes were biologically plausible. In summary, this systematic review provides a critical systematic assessment and an updated elaboration, and its shortcomings highlight the need for further investigation in the field of hematology.
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Adrenérgicos , Hemostasia , Receptores Adrenérgicos , Catecolaminas , Receptores Adrenérgicos/metabolismo , Adrenérgicos/uso terapêutico , Hemostasia/efeitos dos fármacos , Humanos , Estresse Fisiológico , Coagulação SanguíneaRESUMO
Dexmedetomidine is an adrenergic receptor agonist that has been regarded as neuroprotective in several studies without an objective measure to it. Thus, the aim of this meta-analysis was to analyze and quantify the current evidence for the neuroprotective effects of dexmedetomidine in animals. The search was performed by querying the National Library of Medicine. Studies were included based on their language, significancy of their results, and complete availability of data on animal characteristics and interventions. Risk of bias was assessed using SYRCLE's risk of bias tool and certainty was assessed using the ARRIVE Guidelines 2.0. Synthesis was performed by calculating pooled standardized mean difference and presented in forest plots and tables. The number of eligible records included per outcome is the following: 22 for IL-1ß, 13 for IL-6, 19 for apoptosis, 7 for oxidative stress, 7 for Escape Latency, and 4 for Platform Crossings. At the cellular level, dexmedetomidine was found protective against production of IL-1ß (standardized mean difference (SMD) = - 4.3 [- 4.8; - 3.7]) and IL-6 (SMD = - 5.6 [- 6.7; - 4.6]), apoptosis (measured through TUNEL, SMD = - 6.0 [- 6.8; - 4.6]), and oxidative stress (measured as MDA production, SMD = - 2.0 [- 2.4; - 1.4]) exclusively in the central nervous system. At the organism level, dexmedetomidine improved behavioral outcomes measuring escape latency (SMD = - 2.4 [- 3.3; - 1.6]) and number of platform crossings (SMD = 9.1 [- 6.8; - 11.5]). No eligible study had high risk of bias and certainty was satisfactory for reproducibility in all cases. This meta-analysis highlights the complexity of adrenergic stimulation and sheds light into the mechanisms potentiated by dexmedetomidine, which could be exploited for improving current neuroprotective formulations.
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Dexmedetomidina , Fármacos Neuroprotetores , Estados Unidos , Interleucina-6 , Reprodutibilidade dos TestesRESUMO
The organization of a circadian system includes an endogenous pacemaker system, input pathways for environmental synchronizing (entraining) stimuli, and output pathways through which the clock regulates physiological and behavioral processes, for example, the glucose-sensing mechanism in the liver. The liver is the central regulator of metabolism and one of our peripherals clocks. In mammals, central to this pacemaker are the transcription factors Circadian Locomotor Output Cycles Kaput (CLOCK) and BMAL1 (Brain and Muscle ARNT-Like 1). BMAL1 dimerizes with CLOCK, and this heterodimer then binds to the E-box promoter elements (CACGTG) present in clock and clock-controlled genes (CCGs). However, we are just beginning to understand how output pathways and regulatory mechanisms of CCGs are involved in rhythmic physiological processes. Glucokinase (GCK) is a fundamental enzyme in glucose homeostasis, catalyzing the high Km phosphorylation of glucose and allowing its storage. Moreover, gck is a dependent circadian gene. This study aims to determine the contribution of clock genes to hepatic gck expression and to define the specific role of E-box sequences on the circadian regulation of hepatic gck. Results showed that gck expression follows a circadian rhythm in rat hepatocytes in vitro. Accordingly, bmal1 expression induces the glucokinase circadian rhythmic expression in hepatocytes and the analysis of human and rat gck promoters, indicating the presence of E-box regions. Moreover, the basal activity of gck promoter was increased by clock/bmal1 co-transfection but inhibited by Period1/Period2 (per1/per2) co-transfection. Thus, the data suggest that the clock proteins tightly regulate the transcriptional activity of the gck promoter.
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Fatores de Transcrição ARNTL , Elementos E-Box , Ratos , Humanos , Animais , Fatores de Transcrição ARNTL/genética , Glucoquinase , Ritmo Circadiano/fisiologia , Glucose , Regulação da Expressão Gênica , Mamíferos/genéticaRESUMO
Sepsis syndrome develops through enhanced secretion of pro-inflammatory cytokines and the generation of reactive oxygen species (ROS). Sepsis syndrome is characterized by vascular hyperpermeability, hypotension, multiple organ dysfunction syndrome (MODS), and increased mortality, among others. Endotoxemia-derived sepsis is an important cause of sepsis syndrome. During endotoxemia, circulating endotoxin interacts with endothelial cells (ECs), inducing detrimental effects on endothelium function. The endotoxin induces the conversion of ECs into fibroblasts, which are characterized by a massive change in the endothelial gene-expression pattern. This downregulates the endothelial markers and upregulates fibrotic proteins, mesenchymal transcription factors, and extracellular matrix proteins, producing endothelial fibrosis. Sepsis progression is modulated by the consumption of specific nutrients, including ω-3 fatty acids, ascorbic acid, and polyphenolic antioxidant flavonoids. However, the underlying mechanism is poorly described. The notion that gene expression is modulated during inflammatory conditions by nutrient consumption has been reported. However, it is not known whether nutrient consumption modulates the fibrotic endothelial gene-expression pattern during sepsis as a mechanism to decrease vascular hyperpermeability, hypotension, MODS, and mortality. Therefore, the aim of this study was to investigate the impact of the consumption of dietary ω-3 fatty acids, ascorbic acid, and polyphenolic antioxidant flavonoid supplements on the modulation of fibrotic endothelial gene-expression patterns during sepsis and to determine the effects on sepsis outcomes. Our results indicate that the consumption of supplements based on ω-3 fatty acids and polyphenolic antioxidant flavonoids was effective for improving endotoxemia outcomes through prophylactic ingestion and therapeutic usage. Thus, our findings indicated that specific nutrient consumption improves sepsis outcomes and should be considered in treatment.
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Background: There is abundant ethnopharmacological evidence the uses of regarding Solanum species as antitumor and anticancer agents. Glycoalkaloids are among the molecules with antiproliferative activity reported in these species. Purpose: To evaluate the anticancer effect of the Solanum glycoalkaloid tomatine in hepatocellular carcinoma (HCC) in vitro (HepG2 cells) and in vivo models. Methods: The resazurin reduction assay was performed to detect the effect of tomatine on cell viability in human HepG2 cell lines. Programmed cell death was investigated by means of cellular apoptosis assays using Annexin V. The expression of cancer related proteins was detected by Western blotting (WB). Reactive oxygen species (ROS) and calcium were determined by 2,7-dichlorodihydrofluorescein diacetate and Fluo-4, respectively. Intrahepatic HepG2 xenograft mouse model was used to elucidate the effect of tomatine on tumor growth in vivo. Results and Discussion: Tomatine reduced HepG2 cell viability and induced the early apoptosis phase of cell death, consistently with caspase-3, -7, Bcl-2 family, and P53 proteins activation. Furthermore, tomatine increased intracellular ROS and cytosolic Ca+2 levels. Moreover, the NSG mouse xenograft model showed that treating mice with tomatine inhibited HepG2 tumor growth. Conclusion: Tomatine inhibits in vitro and in vivo HCC tumorigenesis in part via modulation of p53, Ca+2, and ROS signalling. Thus, the results suggest the potential cancer therapeutic use of tomatine in HCC patients.
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The combination of biomaterials and stem cells for clinical applications constitute a great challenge in bone tissue engineering. Hence, cellular networks derived from cells-biomaterials crosstalk have a profound influence on cell behaviour and communication, preceding proliferation and differentiation. The purpose of this study was to investigate in vitro cellular networks derived from human gingival mesenchymal stem cells (hGMSCs) and calcium phosphate (CaP) bioceramic interaction. Biological performance of CaP bioceramic and hGMSCs interaction was evaluated through cell adhesion and distribution, cellular proliferation, and potential osteogenic differentiation, at three different times: 5 h, 1 week and 4 weeks. Results confirmed that hGMSCs met the required MSCs criteria while displaying osteogenic differentiaton capacities. We found a significant increase of cellular numbers and proliferation levels. Also, protein and mRNA OPN expression were upregulated in cells cultured with CaP bioceramic by day 21, suggesting an osteoinductible effect of the CaP bioceramic on hGMSCs. Remarkably, CaP bioceramic aggregations were obtained through hGMSCs bridges, suggesting the in vitro potential of macrostructures formation. We conclude that hGMSCs and CaP bioceramics with micro and macropores support hGMSC adhesion, proliferation and osteogenic differentiation. Our results suggest that investigations focused on the interface cells-biomaterials are essential for bone tissue regenerative therapies.
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Materiais Biocompatíveis/farmacologia , Regeneração Óssea/efeitos dos fármacos , Fosfatos de Cálcio/farmacologia , Comunicação Celular/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Osteogênese/efeitos dos fármacos , Engenharia Tecidual/métodosRESUMO
In recent years, the vascular endothelium has gained attention as a key player in the initiation and development of pregnancy disorders. Endothelium acts as an endocrine organ that preserves the homeostatic balance by responding to changes in metabolic status. However, in metabolic disorders, endothelial cells adopt a dysfunctional function, losing their normal responsiveness. During pregnancy, several metabolic changes occur, in which endothelial function decisively participates. Similarly, when pregnancy metabolic disorders occur, endothelial dysfunction plays a key role in pathogenesis. This review outlines the main findings regarding endothelial dysfunction in three main metabolic pathological conditions observed during pregnancy: gestational diabetes, hypertensive disorders, and obesity and hyperlipidemia. Organ, histological and cellular characteristics were thoroughly described. Also, we focused in discussing the underlying molecular mechanisms involved in the cellular signaling pathways that mediate responses in these pathological conditions.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Doenças Metabólicas/metabolismo , Complicações na Gravidez/metabolismo , Animais , Diabetes Gestacional/metabolismo , Eclampsia/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/patologia , Feminino , Homeostase , Humanos , Hiperlipidemias/complicações , Hiperlipidemias/metabolismo , Hipertensão Induzida pela Gravidez/metabolismo , Lipídeos/sangue , Doenças Metabólicas/complicações , Doenças Metabólicas/genética , Obesidade Materna/complicações , Obesidade Materna/metabolismo , Pré-Eclâmpsia/metabolismo , Gravidez , Complicações na Gravidez/genética , Complicações na Gravidez/patologia , Transdução de SinaisRESUMO
BACKGROUND: Main pathological features detected during sepsis and endotoxemia include over-secretion of pro-inflammatory cytokines and multiorgan dysfunction syndrome (MODS). Unfortunately, current clinical efforts to treat sepsis are unsatisfactory, and mortality remains high. Interestingly, transient receptor potential (TRP) melastatin 7 (TRPM7) ion channel controlling Ca2+ and Mg2+ permeability is involved in cytokine production and inflammatory response. Furthermore, TRPM7 downregulation has been shown to alleviate local symptoms in some models of sepsis, but its effects at a systemic level remain to be explored. OBJECTIVE: To test whether TRPM7 mediates cytokine production and MODS during endotoxemia. METHODS: Endotoxemic and sham-endotoxemic rats were subjected to pharmacological inhibition of TRPM7 using carvacrol, or to expression suppression by adenovirus delivery of shRNA (AdVshTRPM7). Then, cytokine and MODS levels in the blood were measured. RESULTS: Inhibition of TRPM7 with carvacrol and suppression with AdVshTRPM7 were both efficient in inhibiting the over-secretion of pro-inflammatory cytokines TNF-α, IL-1ß, IL-6, and IL-12, in endotoxemic rats, without inducing downregulation in blood levels of antiinflammatory cytokines IL-10 and IL-4. Additionally, the use of carvacrol and AdVshTRPM7 significantly prevented liver and pancreas dysfunction, altered metabolic function, and hypoglycemia, induced by endotoxemia. Furthermore, muscle mass wasting and cardiac muscle damage were also significantly reduced by the use of carvacrol and AdVshTRPM7 in endotoxemic rats. CONCLUSION: Our results indicate TRPM7 ion channel as a key protein regulating inflammatory responses and MODS during sepsis. Moreover, TRPM7 appears as a novel molecular target for the management of sepsis.
Assuntos
Cimenos/uso terapêutico , Síndrome da Liberação de Citocina/prevenção & controle , Citocinas/biossíntese , Endotoxemia/complicações , Vetores Genéticos/uso terapêutico , Insuficiência de Múltiplos Órgãos/prevenção & controle , Interferência de RNA , RNA Interferente Pequeno/uso terapêutico , Canais de Cátion TRPM/antagonistas & inibidores , Adenoviridae/genética , Animais , Caquexia/etiologia , Caquexia/prevenção & controle , Cimenos/farmacologia , Síndrome da Liberação de Citocina/etiologia , Vetores Genéticos/genética , Hipoglicemia/etiologia , Hipoglicemia/prevenção & controle , Falência Hepática/etiologia , Falência Hepática/prevenção & controle , Insuficiência de Múltiplos Órgãos/etiologia , Insuficiência de Múltiplos Órgãos/patologia , Músculo Esquelético/patologia , Miocárdio/patologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Ratos , Canais de Cátion TRPM/biossíntese , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/fisiologiaRESUMO
Hypersaline environments represent some of the most challenging settings for life on Earth. Extremely halophilic microorganisms have been selected to colonize and thrive in these extreme environments by virtue of a broad spectrum of adaptations to counter high salinity and osmotic stress. Although there is substantial data on microbial taxonomic diversity in these challenging ecosystems and their primary osmoadaptation mechanisms, less is known about how hypersaline environments shape the genomes of microbial inhabitants at the functional level. In this study, we analyzed the microbial communities in five ponds along the discontinuous salinity gradient from brackish to salt-saturated environments and sequenced the metagenome of the salt (halite) precipitation pond in the artisanal Cáhuil Solar Saltern system. We combined field measurements with spectrophotometric pigment analysis and flow cytometry to characterize the microbial ecology of the pond ecosystems, including primary producers and applied metagenomic sequencing for analysis of archaeal and bacterial taxonomic diversity of the salt crystallizer harvest pond. Comparative metagenomic analysis of the Cáhuil salt crystallizer pond against microbial communities from other salt-saturated aquatic environments revealed a dominance of the archaeal genus Halorubrum and showed an unexpectedly low abundance of Haloquadratum in the Cáhuil system. Functional comparison of 26 hypersaline microbial metagenomes revealed a high proportion of sequences associated with nucleotide excision repair, helicases, replication and restriction-methylation systems in all of them. Moreover, we found distinctive functional signatures between the microbial communities from salt-saturated (>30% [w/v] total salinity) compared to sub-saturated hypersaline environments mainly due to a higher representation of sequences related to replication, recombination and DNA repair in the former. The current study expands our understanding of the diversity and distribution of halophilic microbial populations inhabiting salt-saturated habitats and the functional attributes that sustain them.
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In orthopedic medicine, a feasible reconstruction of bone structures remains one of the main challenges both for healthcare and for improvement of patients' quality of life. There is a growing interest in mesenchymal stem cells (MSCs) medical application, due to their multilineage differentiation potential, and tissue engineering integration to improve bone repair and regeneration. In this review we will describe the main characteristics of MSCs, such as osteogenesis, immunomodulation and antibacterial properties, key parameters to consider during bone repair strategies. Moreover, we describe the properties of calcium phosphate (CaP) bioceramics, which demonstrate to be useful tools in combination with MSCs, due to their biocompatibility, osseointegration and osteoconduction for bone repair and regeneration. Also, we overview the main characteristics of dental cavity MSCs, which are promising candidates, in combination with CaP bioceramics, for bone regeneration and tissue engineering. The understanding of MSCs biology and their interaction with CaP bioceramics and other biomaterials is critical for orthopedic surgical bone replacement, reconstruction and regeneration, which is an integrative and dynamic medical, scientific and bioengineering field of research and biotechnology.
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Regeneração Óssea , Fosfatos de Cálcio/química , Cerâmica/química , Células-Tronco Mesenquimais/citologia , Diferenciação Celular , Humanos , Osteogênese , Engenharia TecidualRESUMO
Transforming growth factor-ß1 (TGF-ß1) potently induces the epithelial-mesenchymal transition (EMT) during tumoral progression. Although Sky-interacting protein (SKIP) regulates TGF-ß1-induced Smad activation, its role in the induction of cell malignance remains uncertain. We found that TGF-ß1 increases SKIP expression in PDV cells. In cells stably transfected with SKIP antisense, AS-S, Smad3 activation decreased, along with an inhibition of TGF-ß1-induced EMT, and the cells were sensitized to the TGF-ß1-dependent inhibition of proliferation. Also, AS-S cells showed a weaker migration and invasion response. Moreover, TGF-ß1-induced urokinase-type plasminogen activator expression was inhibited, concomitantly with a TGF-ß1-independent increment of the plasminogen-activator inhibitor-1 expression. Thus, these results suggest that SKIP is required for EMT and invasiveness induced by TGF-ß1 in transformed cells.
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Movimento Celular/efeitos dos fármacos , Transformação Celular Neoplásica , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , Invasividade Neoplásica , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Inibidor 1 de Ativador de Plasminogênio/genética , Fatores de Transcrição , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Transforming growth factor-beta1 (TGF-beta1) activates Rac1 GTPase in mouse transformed keratinocytes. Expression of a constitutively active Q61LRac1 mutant induced an epithelial to mesenchymal transition (EMT) linked to stimulation of cell migration and invasion. On the contrary, expression of a dominant-negative N17TRac1 abolished TGF-beta1-induced cell scattering, migration and invasion. Moreover, Q61LRac1 enhanced metalloproteinase-9 (MMP9) production to levels comparable to those induced by TGF-beta1, while N17TRac1 was inhibitory. TGF-beta1-mediated EMT involves the expression of the E-cadherin repressor Snail1, regulated by the Rac1 and mitogen-activated protein kinase (MAPK) pathways. Furthermore, MMP9 production was MAPK-dependent, as the MEK inhibitor PD98059 decreased TGF-beta1-induced MMP9 expression and secretion in Q61LRac1 expressing cells. We propose that regulation of TGF-beta1-mediated plasticity of transformed keratinocytes requires the cooperation between the Rac1 and MAPK signalling pathways.
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Células Epiteliais/metabolismo , Queratinócitos/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Caderinas/metabolismo , Desdiferenciação Celular , Movimento Celular , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de SinaisRESUMO
TGF-beta1 has been postulated as a pro-oncogenic factor in the late step of the tumoral progression. In transformed cells, TGF-beta1 enhances the capacity to degrade the extracellular matrix, cell invasiveness and epithelial-mesenchymal transition, which are crucial steps for metastasis. Urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) are critical components in cell migration and invasion induced by TGF-beta1, however, the exact mechanism by which TGF-beta1 regulates uPA and MMP-9 is not well elucidated so far. In the present study, we analyzed the role of ROS-NFkappaB, signal as mediator in the cell malignity enhancement by TGF-beta1. We found that TGF-beta1 activates NFkappaB, through Rac1-NOXs-ROS-dependent mechanism. Our results shows that TGF-beta1 stimulation of uPA and MMP-9 expression involve NOXs-dependent ROS and NFkappaB, activation, demonstrated by using DPI, NOXs inhibitor, ROS scavenger N-acetylcysteine and SN50, an NFkb inhibitor. Furthermore, we found that the inhibition of ROS and NFkappaB, abrogates TGF-beta1 stimulation of EMT, cell motility and invasion. Thus, ROS-NFkappaB acts as the crucial signal in TGF-beta1-induced uPA and MMP-9 expression thereby mediating the enhancement of cellular malignity by TGF-beta1.
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Movimento Celular , Queratinócitos/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição RelA/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular Transformada , Movimento Celular/efeitos dos fármacos , Transdiferenciação Celular , Inibidores Enzimáticos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Genes Reporter , Queratinócitos/efeitos dos fármacos , Metaloproteinase 9 da Matriz/genética , Camundongos , Mutação , NADPH Oxidases/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Transdução de Sinais , Fatores de Tempo , Fator de Transcrição RelA/genética , Transfecção , Regulação para Cima , Ativador de Plasminogênio Tipo Uroquinase/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
TGF-beta1 is a potent inductor of malignance in cancer cells. TGF-beta1 stimulates the expression of extracellular matrix degrading proteases, cell migration and it is also involved in the epithelial-mesenchymal transition (EMT). In the present work, we analyzed the role of Spred2 in the urokinase-type plasminogen activator (uPA) stimulation, EMT and cell migration by TGF-beta1. We found that both the expression of mRNA and the protein level of Spred2 were lower in transformed keratinocytes PDV compared with immortalized keratinocytes MCA-3D. The transient ectopic expression of Spred2 in PDV cells inhibited the TGF-beta1-transactivated SRE-Luc reporter which is related with the ERK1,2 signal. The stable ectopic expression of Spred2 in PDV cells (SP cells) led to the loss of ERK 1,2 activation by TGF-beta1, although Smad2 activation was not affected, and the knockdown of Spred2 enhanced the activation of ERK1,2 signal by TGF-beta1. The increment of uPA expression induced by TGF-beta1 was suppressed in SP cells. In contrast, the stimulus on PAI-1 expression was not affected and comparable to parental PDV cells. SP cells under TGF-beta1 treatment were unable to display the EMT, since the overexpression of Spred2 abolished the TGF-beta1-induced disruption of the E-cadherin cell to cell interactions, reorganization of the actin cytoskeleton and upregulation of the mesenchymal marker vimentin. Finally, SP cells could not respond to the TGF-beta1 stimulus on cell migration. Taken together, the data in the present study suggests that Spred2 is a regulator of TGF-beta1-induced malignance in transformed keratinocytes.
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Movimento Celular/fisiologia , Células Epiteliais/citologia , Mesoderma/citologia , Proteínas Repressoras/fisiologia , Fator de Crescimento Transformador beta1/fisiologia , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Sequência de Bases , Primers do DNA , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Imunofluorescência , Técnicas de Silenciamento de Genes , Humanos , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In the present study, we demonstrated that changes in Rac1 activity associated with the production of intracellular ROS modulate the migratory properties in MCF-7 and T47D human mammary cell lines. We also described that the NFkappaB pathway exerts a downstream control on the expression of the ROS-dependent cellular migratory potential. These results emphasize the importance of redox balance in the acquisition of malignancy and support previous data sustaining that an oxidative environment predisposes cells to activate signal-transduction pathways actively involved in cellular oncogenesis. Our data also provides evidence that NADPH oxidase could constitute the main source of intracellular ROS in response to changes in Rac1 activity. We suggest that Rac1 plays a role in cellular migration not only limited to its known function in reorganization of the actin cytoskeleton, but also as part of the intracellular machinery that controls the redox balance.
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Movimento Celular/efeitos dos fármacos , NADPH Oxidases/metabolismo , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Linhagem Celular , Humanos , Transdução de SinaisRESUMO
In this study we analyzed the role of the c-Jun N-terminal kinases (JNK) pathway in the TGF-beta1 stimulation of urokinase-type plasminogen activator (uPA), initial stages of epithelial-mesenchymal transdifferentiation (EMT) and cell migration. TGF-beta1 induces JNK phosphorylation, c-Jun transactivation and AP1 activation. The involvement of JNK was evaluated using dominant negative mutants SEK-1 AL, JNK and cJun, depletion of JNK1,2 proteins by treatment of cells with antisense oligonucleotides, as well as the chemical inhibitor SP600125. Our results demonstrated that the JNK pathway is required in the TGF-beta1 enhancement of uPA, fibronectin, E-cadherin delocalization, actin re-organization and vimentin expression, concomitant with the induction of cell migration. These results allow us to suggest a role of JNK in the TGF-beta1 induction of EMT in relation with the stimulation of malignant properties of mouse transformed keratinocytes.
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Diferenciação Celular/efeitos dos fármacos , Queratinócitos/enzimologia , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Animais , Caderinas/genética , Caderinas/metabolismo , Diferenciação Celular/genética , Linhagem Celular Transformada , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Fibronectinas/biossíntese , Fibronectinas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Queratinócitos/patologia , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Camundongos , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais/genética , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1 , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Ativador de Plasminogênio Tipo Uroquinase/genética , Vimentina/biossíntese , Vimentina/genéticaRESUMO
Efforts have been made to develop a chemoprevention that selectively triggers apoptosis in malignant cancer cells. Here, we demonstrated that a mutated Ha-Ras activity is required in Anisomycin-induced apoptosis in transformed keratinocytes. Anisomycin stimulates JNK activity and apoptosis in oncogenic Ha-Ras positive cells, but not in normal keratinocytes. This effect was demonstrated in stably transfected cells with dominant negative Ha-Ras, that protected transformed cells, and oncogenic Ha-Ras that sensitized non-transformed cells to Anisomycin-induced apoptosis. Lastly, the treatment of cells with inhibitors of the JNK displayed resistance to Anisomycin induced apoptosis. These data suggests that the oncogenic Ha-Ras is important for Anisomycin-induced JNK activation and apoptosis in transformed keratinocytes.
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Anisomicina/farmacologia , Apoptose/genética , Genes ras/genética , Queratinócitos/efeitos dos fármacos , Proteína Oncogênica p21(ras)/genética , Animais , Linhagem Celular Transformada , Ativação Enzimática , Queratinócitos/metabolismo , MAP Quinase Quinase 4/metabolismo , Camundongos , Mutação , Proteína Oncogênica p21(ras)/metabolismo , Pele/citologiaRESUMO
BACKGROUND: Regulation of the extracellular matrix turnover is a crucial process in wound healing and the progress of periodontal disease. It has been proposed that urokinase-type plasminogen activator (uPA), under the control of growth factors or cytokines, provides the proteolytic potential to the accomplishment of these cellular events. Epidermal growth factor (EGF) is one of the growth factors that has been shown to be active in uPA regulation. METHODS: In this study, we have assessed the effect of EGF on uPA expression in primary cultures of human gingival fibroblasts. We also studied the signaling pathways involved in this process and the role of the dietary phytoestrogens curcumin and genistein as potential modulators of this response. RESULTS: Human gingival fibroblasts expressed a basal uPA activity, which was inhibited by genistein, but not by curcumin. After treatment with 10 ng/ml EGF, uPA production was strongly stimulated. Exposure to genistein and curcumin inhibited EGF-stimulated urokinase production, although only genistein showed a statistically significant inhibitory response. Using more specific inhibitors, we found that the mitogen-activated extracellular kinase and c-Jun N-terminal kinase (JNK) inhibitors PD98059 and SP600125 also blocked the EGF-dependent stimulatory effect. On the other hand, SB203580, inhibitor of the p38 member of mitogen-activated protein kinase family, did not alter this response. In accordance to these findings, EGF stimulated a potent activation of JNK and a mild activation of extracellular signal-regulated kinases 1/2. Finally, EGF stimulated the phosphorylation of its receptor and tyrphostin (AG1478), curcumin and genistein were able to inhibit this stimulatory effect. CONCLUSIONS: These results indicate that EGF constitutes a strong stimuli on uPA expression in human gingival fibroblasts. Our data also shows that EGF-stimulated uPA production involves the activation of the extracellular signal-regulated kinases 1/2 and JNK signaling pathways and might be modulated by the natural phytoestrogens curcumin and genistein.
Assuntos
Curcumina/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Genisteína/farmacologia , Gengiva/enzimologia , Fitoestrógenos/farmacologia , Ativadores de Plasminogênio/biossíntese , Inibidores de Proteínas Quinases/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Fator de Crescimento Epidérmico/fisiologia , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Fibroblastos/enzimologia , Expressão Gênica/efeitos dos fármacos , Gengiva/citologia , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ativadores de Plasminogênio/antagonistas & inibidores , Ativadores de Plasminogênio/genética , Inibidores de Proteases , Proteínas Recombinantes/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Mouse-transformed keratinocytes cultured in the presence of transforming growth factor beta1 (TGF-beta1) acquire an array of morphologic and functional properties that give rise to a migratory phenotype that expresses mesenchymal molecular markers. This cellular conversion involves activation of the Ras-ERK pathway, enhancement of urokinase (uPA) and matrix metalloproteinase-9 (MMP-9) expression and induction of invasiveness. In our present work, we demonstrate that cAMP and forskolin are able to prevent the expression of these mesenchymal properties, probably due to blockade of the Ras-ERK pathway. Our results also show that cAMP and forskolin are able to abolish the TGF-beta1-induced reorganization of the actin cytoskeleton that is characteristic of the mesenchymal phenotype and also inhibits the disruption of the E-cadherin cell to cell interactions. The latter responses seem to depend on the activity of protein kinase A, as demonstrated by the activation of the Ras-ERK pathway by specific protein kinase A inhibitors.