RESUMO
Melatonin and its indoles derivatives are central in the synchronization of malaria parasites. In this research, we discovered that melatonin is unable to increase the parasitemia in the human malaria Plasmodium falciparum that lacks the kinase PfeIK1. The PfeIK1 knockout strain is a valuable tool in the screening of indol-related compound that blocks the melatonin effect in wild-type (WT) parasite development. The assays were performed by using flow cytometry with simultaneous labeling for mitochondria viability with MitoTracker Deep Red and nucleus staining with SYBR Green. We found that Melatotosil leads to an increase in parasitemia in P. falciparum and blocks melatonin effect in the WT parasite. Using microscopy imaging system, we found that Melatotosil at 500 nM is able to induce cytosolic calcium rise in transgenic PfGCaMP3 parasites. On the contrary, the compound Triptiofen blocks P. falciparum cell cycle with IC50 9.76 µM ± 0.6, inhibits melatonin action, and does not lead to a cytosolic calcium rise in PfGCaMP3 parasites. We also found that the synthetic indol-related compounds arrested parasite cycle for PfeIK1 knockout and (WT) P. falciparum (3D7) in 72 hours culture assays with the IC50 values slighting lower for the WT strain. We concluded that the kinase PfeIK1 is central for melatonin downstream signaling pathways involved in parasite cell cycle progression. More importantly, the indol-related compounds block its cycle as an upstream essential mechanism for parasite survival. Our data clearly show that this class of compounds emerge as an alternative for the problem of resistance with the classical antimalarials.
Assuntos
Antimaláricos/farmacologia , Ciclo Celular , Malária Falciparum/enzimologia , Plasmodium falciparum/enzimologia , Transdução de Sinais , Proteínas Elk-1 do Domínio ets/antagonistas & inibidores , Antimaláricos/química , Humanos , Malária Falciparum/tratamento farmacológico , Melatonina , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
Voltage-gated Ca2+ (CaV) channels are expressed in endocrine cells where they contribute to hormone secretion. Diverse chemical messengers, including epidermal growth factor (EGF), are known to affect the expression of CaV channels. Previous studies have shown that EGF increases Ca2+ currents in GH3 pituitary cells by increasing the number of high voltage-activated (HVA) CaV channels at the cell membrane, which results in enhanced prolactin (PRL) secretion. However, little is known regarding the mechanisms underlying this regulation. Here, we show that EGF actually increases the expression of the CaVα2δ-1 subunit, a key molecular component of HVA channels. The analysis of the gene promoter encoding CaVα2δ-1 (CACNA2D1) revealed binding sites for transcription factors activated by the Ras/Raf/MEK/ERK signaling cascade. Chromatin immunoprecipitation and site-directed mutagenesis showed that ELK-1 is crucial for the transcriptional regulation of CACNA2D1 in response to EGF. Furthermore, we found that EGF increases the membrane expression of CaVα2δ-1 and that ELK-1 overexpression increases HVA current density, whereas ELK-1 knockdown decreases the functional expression of the channels. Hormone release assays revealed that CaVα2δ-1 overexpression increases PRL secretion. These results suggest a mechanism for how EGF, by activating the Ras/Raf/MEK/ERK/ELK-1 pathway, may influence the expression of HVA channels and the secretory behavior of pituitary cells.
Assuntos
Canais de Cálcio Tipo L/genética , Fator de Crescimento Epidérmico/metabolismo , Regulação da Expressão Gênica , Sistema de Sinalização das MAP Quinases/genética , Proteínas Elk-1 do Domínio ets/genética , Quinases raf/genética , Proteínas ras/genética , Animais , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Técnicas de Silenciamento de Genes , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Ratos , Transdução de Sinais , Proteínas Elk-1 do Domínio ets/metabolismo , Quinases raf/metabolismo , Proteínas ras/metabolismoRESUMO
Expression of the estrogen receptor ESR1 is higher in the corpus than it is in the initial segment/caput and cauda of the epididymis. ESR1 immunostaining in the corpus has been localized not only in the nuclei but also in the cytoplasm and apical membrane, which indicates that ESR1 plays a role in membrane-initiated signaling. The present study investigated whether ESR1 mediates the activation of rapid signaling pathways by estradiol (E2) in the epididymis. We investigated the effect of E2 and the ESR1-selective agonist (4,4',4''-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol (PPT) on the activation of extracellular signal-regulated protein kinases (ERK1/2), CREB protein, and ETS oncogene-related protein (ELK1). Treatment with PPT did not affect ERK1/2 phosphorylation in the cauda, but it rapidly increased ERK1/2 phosphorylation in the initial segment/caput and corpus of the epididymis. PPT also activated CREB and ELK1 in the corpus of the epididymis. The PPT-induced phosphorylation of ERK1/2, CREB, and ELK1 was blocked by the ESR1-selective antagonist MPP and by pretreatment with a non-receptor tyrosine kinase SRC inhibitor, an EGFR kinase inhibitor, an MEK1/2 inhibitor, and a phosphatidylinositol-3-kinase inhibitor. In conclusion, these results indicate that the corpus, which is a region with high expression of the estrogen receptor ESR1, is a major target in the epididymis for the activation of rapid signaling by E2. The sequence of events that follow E2 interaction with ESR1 includes the SRC-mediated transactivation of EGFR and the phosphorylation of ERK1/2, CREB, and ELK1. This rapid estrogen signaling may modulate gene expression in the corpus of the epididymis, and it may play a role in the dynamic microenvironment of the epididymal lumen.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Epididimo/enzimologia , Receptor alfa de Estrogênio/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismo , Animais , Estradiol/fisiologia , Receptor alfa de Estrogênio/agonistas , Sistema de Sinalização das MAP Quinases , Masculino , Fenóis/farmacologia , Fosforilação , Processamento de Proteína Pós-Traducional , Pirazóis/farmacologia , Ratos WistarRESUMO
OBJETIVO: Estimar a prevalência de dor crônica e sua associação com a situação socioeconômica, demográfica e atividade física no lazer em idosos. MÉTODOS: Este estudo é parte do inquérito epidemiológico e transversal de base populacional e domiciliar EpiFloripa Idoso 2009-2010 realizado com 1.705 idosos (≥ 60 anos), residentes em Florianópolis, Santa Catarina. A partir da resposta afirmativa de dor crônica, foram investigadas as associações com as variáveis obtidas por meio de entrevista estruturada. Realizou-se a estatística descritiva, incluindo cálculos de proporções e intervalos de confiança 95% (IC95%). Na análise bruta e ajustada, empregou-se regressão de Poisson, estimando-se as razões de prevalência, com intervalos de confiança de 95% e valores p ≤ 0,05. RESULTADOS: Dentre os idosos investigados, 29,3% (IC95% 26,5 - 32,2) relataram dor crônica. Na análise ajustada, observou-se que as variáveis sexo feminino, menor escolaridade e pior situação econômica ficaram associadas significativamente com maior prevalência de dor crônica; ser fisicamente ativo no lazer ficou associado significativamente com menor prevalência do desfecho. CONCLUSÕES: Percebe-se que a dor crônica é um agravo que acomete considerável parcela de idosos, havendo desigualdades sociais na sua frequência e sendo beneficamente afetada pela atividade física no lazer. É necessário que políticas públicas de saúde subsidiem programas multidisciplinares de controle da dor incluindo a prática regular de atividade física, voltada especificamente à promoção da saúde do idoso, evitando assim que a dor crônica comprometa a qualidade de vida desta população. .
OBJECTIVE: To estimate the prevalence of chronic pain and its association with socioeconomic and demographic status, and leisure physical activity in the elderly population. METHODS: This study is part of an epidemiological cross-sectional population-based household survey called EpiFloripa Elderly 2009-2010, which was conducted with 1,705 elderly individuals (≥ 60 years) residents of Florianópolis, Santa Catarina. From the positive response to chronic pain, the associations with the variables were investigated through a structured interview. Descriptive statistics were conducted, including ratio calculation and 95% confidence intervals. In crude and adjusted analysis, Poisson regression was utilized, estimating prevalence ratios, with 95% confidence intervals and ≤ 0.05 p-values. RESULTS: Among the subjects, 29.3% (IC95% 26.5 - 32.2) reported chronic pain. Adjusted analysis showed that being female, having less years of schooling, and being in worse economic situation were significantly associated with a higher prevalence of chronic pain. Being physically active during leisure time was significantly associated with lower prevalence of the outcome. CONCLUSIONS: Therefore, it is clear that chronic pain affects a considerable amount of elderly individuals. Social inequalities are a harmful influence in these individuals' quality of life, inasmuch as those inequalities increase the frequency with which chronic pain afflicts them. At the same time, physical activity during leisure time decreases chronic pain frequency. It is fundamental that public health policies subsidize multidisciplinary pain management programs, which should include health targeted physical activity for the elderly, thus preventing the decrease in quality of life that chronic pain brings to this population. .
Assuntos
Animais , Humanos , Proteína 1 de Resposta de Crescimento Precoce/genética , Células Epiteliais/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , /metabolismo , Sulindaco/análogos & derivados , Apoptose/efeitos dos fármacos , Western Blotting , Butadienos/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Imidazóis/farmacologia , Intestinos/citologia , Intestinos/efeitos dos fármacos , Intestinos/metabolismo , Luciferases/genética , Luciferases/metabolismo , Microscopia Confocal , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , /antagonistas & inibidores , Nitrilas/farmacologia , Piridinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulindaco/farmacologia , Transfecção , Regulação para Cima/efeitos dos fármacos , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
We have previously reported in C2C12 murine skeletal muscle cells that 10(-8)M 17ß-estradiol promotes MAPKs stimulation which in turn mediates the activation of CREB and Elk-1 transcription factors. In this work, we demonstrated that the hormone induces ERK2 phosphorylation (without affecting ERK1 activation) and also stimulates p38 MAPK, both in a dose-dependent manner. Moreover, estrogen receptors involvement in MAPKs activation by the estrogen was studied. The use of ICI182780 (1 µM), an antagonist of ERs, and specific siRNAs to block ERα and ERß expression, demonstrated that ERα mediates ERK2 activation but not p38 MAPK phosphorylation by 17ß-estradiol, and that ERß isoform is not implicated in MAPKs activation by the hormone. Furthermore, Src and PKC contribution in estrogen stimulation of the MAPKs was investigated. Compounds PP2 and Ro318220, Src and PKC family inhibitors, respectively abrogated ERK2 and p38 MAPK phosphorylation by 17ß-estradiol. Of interest, the hormone was able to induce Src and PKCδ activation. In addition, Ro318220 decreased estrogen-dependent Src modulation implicating PKC in hormone upregulation of Src. Accordingly, PP2 and Ro318220 suppressed CREB and Elk-1 phosphorylation as well as c-Fos and c-Jun oncoprotein levels induced by 17ß-estradiol. Altogether, these data indicate that 17ß-estradiol activates ERK2 through ERα and p38 MAPK in an ERα/ß-independent manner and that PKC and Src proteins are key upstream components on MAPKs activation in C2C12 skeletal muscle cells.
Assuntos
Estradiol/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteína Quinase C/metabolismo , Receptores de Estrogênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases da Família src/metabolismo , Animais , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
Receptors for IgG Abs (Fcgamma receptors) are capable of triggering diverse cell responses in leukocytes. In neutrophils, two Fcgamma receptors, namely FcgammaRIIA and FcgammaRIIIB, are constitutively expressed. The signaling pathways that regulate FcgammaRIIA-mediated phagocytosis have been relatively well described. However, the different signaling pathways that lead to NF activation after engagement of each Fcgamma receptor have only been partially described. To address this problem, neutrophils were stimulated by cross-linking selectively each type of Fcgamma receptor with specific mAbs, and NF activation was then analyzed. FcgammaRIIIB, but not FcgammaRIIA, promoted a robust increase in phosphorylated ERK in the nucleus, and also efficient phosphorylation of the NF Elk-1. Complete mAb 3G8 (anti-FcgammaRIIIB) induced a higher response than did F(ab')(2) fragments of mAb 3G8, suggesting a possible synergistic effect of both FcgammaR receptors. However, mAb IV.3 (anti-FcgammaRIIA) alone did not cause an increase of phosphorylated ERK in the nucleus. FcgammaRIIIB-induced nuclear phosphorylation of ERK, and of Elk-1, was not affected by Syk, PI3K, or MEK inhibitors. In contrast, FcgammaRIIA- or FcgammaRIIIB-mediated phosphorylation of cytoplasmic ERK depended on Syk, PI3K, and MEK. Also, ERK, but not MEK, was constitutively present in the nucleus, and FcgammaRIIIB cross-linking did not increase the levels of nuclear ERK or MEK. These data clearly show that different neutrophil Fcgamma receptors possess different signaling capabilities. FcgammaRIIIB, but not FcgammaRIIA, activates a unique signaling pathway leading to the nuclear-restricted phosphorylation of ERK and Elk-1, independently of Syk, PI3K, or MEK.
Assuntos
Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptores de IgG/imunologia , Receptores de IgG/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismo , Antígenos CD18/farmacologia , Núcleo Celular/metabolismo , Fatores Quimiotáticos/farmacologia , Citoplasma/enzimologia , Citoplasma/imunologia , Humanos , Neutrófilos/efeitos dos fármacos , Fosforilação , Transdução de SinaisRESUMO
S-Nitrosylation reactions are considered to be a major mechanism by which NO-related bioactivities are regulated in vivo. In the present study, we show the effects of the low molecular weight S-nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), on cell cycle progression of rabbit aortic endothelial cells (RAEC). SNAP at low concentrations (0.1mM) stimulated the p21Ras-ERK1/2 MAP kinase signaling pathway. Activation of this signaling pathway was strongly inhibited in cells stably transfected with S-nitrosylation insensitive p21Ras (p21(Ras (C118S))). Furthermore, the SNAP-induced effects on cell cycle progression were eliminated in RAEC expressing N17Ras, a negative dominant mutant of p21Ras. Upon stimulation with SNAP, ERK1/2 MAP kinases become phosphorylated and translocate to the nucleus promoting the phosphorylation of the transcription factor Elk1. Synthesis of Cyclin D1 and stimulation of the cyclin-dependent kinases cdk4 and cdk6 resulted in the phosphorylation of the nuclear protein Rb and its dissociation from the E2F family of transcription factors. Cells then pass the restriction point in the late G1 phase. Cyclins E and A were expressed as the cell cycle progressed through the S phase upon stimulation with SNAP. Further transition in the cell cycle from the G2 to M phase was evidenced by the G2/M peak found in a histogram of the cell-phase distribution in SNAP-treated RAEC. These observations suggest that low molecular weight S-nitrosothiols may promote cell cycle progression possibly through the transnitrosation of p21Ras, and activation of the Ras-ERK1/2 MAP kinases signaling pathway.
Assuntos
Aorta/citologia , Ciclo Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , S-Nitroso-N-Acetilpenicilamina/farmacologia , S-Nitrosotióis/farmacologia , Transporte Ativo do Núcleo Celular , Animais , Células Cultivadas , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Peso Molecular , Fosforilação , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Coelhos , Proteína do Retinoblastoma/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
In mammals, neutrophils are the most abundant circulating leukocytes. Neutrophils are short-lived cells presenting at least two important transcriptionally regulated cellular responses, initiated by cell activation: the production of pro-inflammatory cytokines and the inhibition of apoptosis. The study of transcriptionally regulated processes in these cells cannot be approached through conventional reporter gene strategies, as there are currently not available methods for neutrophil transfection. Here we describe a novel flow cytometry-based method that allowed quantification of nuclear factor NF-kappaB activation in neutrophils, in response to FcgammaIIA and FcgammaRIIIB stimulation. The sensitivity of this method allowed the detection of small changes in NF-kappaB activation, due to pharmacological inhibition of receptor-initiated signaling pathways. NF-kappaB activation was also detected by this method in various leukocyte cell lines. In addition, quantification of Fcgamma receptor-initiated nuclear activation of ERK and Elk-1 was successfully achieved through this method. The broad applicability and versatility of this flow cytometry-based method position it as a fast and reliable alternative to traditional methods for analyzing activation of transcription factors in a variety of cell types.
Assuntos
Antígenos CD/metabolismo , Citometria de Fluxo/métodos , NF-kappa B/sangue , Neutrófilos/metabolismo , Receptores de IgG/metabolismo , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Ligadas por GPI , Humanos , Células Jurkat , NF-kappa B/metabolismo , Neutrófilos/efeitos dos fármacos , Ratos , Transdução de Sinais , Proteínas Elk-1 do Domínio ets/metabolismoRESUMO
The mitogen activated protein kinases (MAPKs) have been classified into at least six subfamilies, among which ERK1/2, JNK1/2 and p38 MAPK are the most extensively studied. The steroid hormones 1alpha,25-dihydroxy-Vitamin D(3) and 17beta-estradiol promote biological responses through activation of MAPK cascades in various cell types. We previously reported that 1alpha,25(OH)(2)D(3) rapidly (within 1 min) activates p38 MAPK in C2C12 skeletal muscle cells. In this work, using the same muscle cell line, we demonstrate that 1alpha,25(OH)(2)D(3) or 17beta-estradiol phosphorylate and activate ERK1/2 and p38 MAPK after longer treatment intervals, maximal effects seen at 90 and 30 min (ERK1/2) and at 60 and 15 min (p38 MAPK) for these hormones, respectively. Hormone-dependent ERK and p38 activation was abolished by MAPK specific inhibitors U0126 and SB203580. 1alpha,25(OH)(2)D(3) and 17beta-estradiol also induced the phosphorylation of CREB and Elk-1 transcription factors in an ERK1/2-dependent manner. Simultaneous addition of both hormones potentiated CREB phosphorylation. 1alpha,25(OH)(2)D(3)- and 17beta-estradiol-induced c-fos expression, which was mediated by p38 phosphorylation. The action of 17beta-estradiol on c-fos levels was also dependent on ERK1/2. These results suggest that MAPK signalling pathways play an important role in regulating early gene expression through CREB and Elk-1 activation in skeletal muscle cells.
Assuntos
Calcitriol/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Estradiol/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Proteínas Elk-1 do Domínio ets/metabolismo , Animais , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Camundongos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transdução de SinaisRESUMO
We have studied the molecular mechanism and signal transduction of pim-1, an oncogene encoding a serine-threonine kinase. This is a true oncogene which prolongs survival and inhibits apoptosis of hematopoietic cells. In order to determine whether the effects of Pim-1 occur by regulation of the mitogen-activated protein kinase pathway, we used a transcriptional reporter assay by transient co-transfection as a screening method. In this study, we found that Pim-1 inhibited the Elk-1 and NFkappaB transcriptional activities induced by activation of the mitogen-activated protein kinase cascade in reporter gene assays. However, Western blots showed that the induction of Elk-1-regulated expression of endogenous c-Fos was not affected by Pim-1. The phosphorylation and activation of neither Erk1/2 nor Elk-1 was influenced by Pim-1. Also, in the gel shift assay, the pattern of endogenous NFkappaB binding to its probe was not changed in any manner by Pim-1. These data indicate that Pim-1 does not regulate the activation of Erk1/2, Elk-1 or NFkappaB. These contrasting results suggest a pitfall of the transient co-transfection reporter assay in analyzing the regulation of transcription factors outside of the chromosome context. It ensures that results from reporter gene expression assay should be verified by study of endogenous gene expression.