RESUMEN
BACKGROUND: Synaptic plasticity is the neuronal capacity to modify the function and structure of dendritic spines (DS) in response to neuromodulators. Sex steroids, particularly 17ß-estradiol (E2) and progesterone (P4), are key regulators in the control of DS formation through multiprotein complexes including WAVE1 protein, and are thus fundamental for the development of learning and memory. OBJECTIVES: The aim of this work was to evaluate the molecular switch Cdk5 kinase/protein phosphatase 2A (PP2A) in the control of WAVE1 protein (phosphorylation/dephosphorylation) and the regulation of WAVE1 and cortactin to the Arp2/3 complex, in response to rapid treatments with E2 and P4 in cortical neuronal cells. RESULTS: Rapid treatment with E2 and P4 modified neuronal morphology and significantly increased the number of DS. This effect was reduced by the use of a Cdk5 inhibitor (Roscovitine). In contrast, inhibition of PP2A with PP2A dominant negative construct significantly increased DS formation, evidencing the participation of kinase/phosphatase in the regulation of WAVE1 in DS formation induced by E2 and P4. Cortactin regulates DS formation via Src and PAK1 kinase induced by E2 and P4. Both cortactin and WAVE1 signal to Arp2/3 complex to synergistically promote actin nucleation. CONCLUSION: These results suggest that E2 and P4 dynamically regulate neuron morphology through nongenomic signaling via cortactin/WAVE1-Arp2/3 complex. The control of these proteins is tightly orchestrated by phosphorylation, where kinases and phosphatases are essential for actin nucleation and, finally, DS formation. This work provides a deeper understanding of the biological actions of sex steroids in the regulation of DS turnover and neuronal plasticity processes.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Corteza Cerebral/fisiología , Espinas Dendríticas/fisiología , Estradiol/fisiología , Progesterona/fisiología , Proteína Fosfatasa 2/metabolismo , Transducción de Señal/fisiología , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/efectos de los fármacos , Animales , Corteza Cerebral/efectos de los fármacos , Cortactina , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Espinas Dendríticas/efectos de los fármacos , Embrión de Mamíferos , Estradiol/farmacología , Progesterona/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteína Fosfatasa 2/efectos de los fármacos , Ratas , Roscovitina/farmacología , Transducción de Señal/efectos de los fármacos , Familia de Proteínas del Síndrome de Wiskott-Aldrich/efectos de los fármacosRESUMEN
BACKGROUND: MicroRNA-7 (miR-7) has been observed as a potent tumour suppressor in multiple cancer types including breast cancer. The aim of this study was to investigate the response sensitivities of metastatic breast cancer cells to miR-7 and the roles of miR-7 in the interaction of endothelial cells and metastatic cancer cells. METHODS: Expression profile of miRNAs in a breast cancer specimen cohort and breast cancer cells were determined using real-time quantitative miRNA assays. Effect of the altering expression of miR-7 on migration, invasion, proliferation, interaction and underlying molecular mechanism of breast cancer cells and endothelial cells was investigated after treatment with the synthesised mimic of miR-7. Luciferase activity analysis was performed to validate Wave-3 as a novel target of miR-7. RESULTS: miR-7 expression was negatively correlated with the stage, grade and survival of the breast cancer patients. There was also differential expression of miRNAs including miR-7 in the breast cancer cells. The synthesised mimic of miR-7 inhibits the motility and wound healing potential of breast cancer cells. The highly metastatic MDA-MB-231 cells are more sensitive to the miR-7 treatment than the poorly invasive MCF-7 cells. Treatment with miR-7 downregulated the expression of EGFR, IGF1R and Wave3 in MDA-MB-231 cells but not in MCF-7 cells. In addition, we further demonstrated that miR-7 inhibited the proliferation, migration and invasion of endothelial cells. And more importantly, miR-7 suppressed the homing and migration of endothelial cells to more aggressive tumour cell conditions. CONCLUSIONS: Given the dual inhibitory effect of miR-7 on metastatic breast cancer cells alone and the interaction of endothelial cells with the tumour-conditioned microenvironment, we suggest miR-7 may be a new therapeutic candidate for its capacity not only to prevent breast cancer cell spreading but also to inhibit tumour-associated angiogenesis in the metastatic breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Células Endoteliales/metabolismo , MicroARNs/genética , Apoptosis , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/genética , Proliferación Celular/genética , Células Endoteliales/efectos de los fármacos , Receptores ErbB/efectos de los fármacos , Receptores ErbB/metabolismo , Femenino , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células MCF-7 , MicroARNs/metabolismo , MicroARNs/farmacología , Metástasis de la Neoplasia , Estadificación de Neoplasias , Pronóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor IGF Tipo 1 , Receptores CXCR/genética , Receptores CXCR4/genética , Receptores de Somatomedina/efectos de los fármacos , Receptores de Somatomedina/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/efectos de los fármacos , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
BACKGROUND: Omega-3 polyunsaturated fatty acids (n-3 PUFAs) show beneficial effects on cardiovascular health and cognitive functions, but the underlying molecular mechanisms are not completely understood. Because of the fact that cytoskeleton dynamics affect almost every cellular process, the regulation of cytoskeletal dynamics could be a new pathway by which n-3 PUFAs exert their effects on cellular level. METHODS: A 12-week open-label intervention study with 12 healthy men was conducted to determine the effects of 2.7 g/d n-3 PUFA on changes in mRNA expression of cytoskeleton-associated genes by quantitative real-time PCR in whole blood. Furthermore, the actin content in red blood cells was analyzed by immunofluorescence imaging. RESULTS: N-3 PUFA supplementation resulted in a significant down-regulation of cytoskeleton-associated genes, in particular three GTPases (RAC1, RHOA, CDC42), three kinases (ROCK1, PAK2, LIMK), two Wiskott-Aldrich syndrome proteins (WASL, WASF2) as well as actin related protein 2/3 complex (ARPC2, ARPC3) and cofilin (CFL1). Variability in F-actin content between subjects was high; reduced actin content was only reduced within group evaluation. CONCLUSIONS: Reduced cytoskeleton-associated gene expression after n-3 PUFA supplementation suggests that regulation of cytoskeleton dynamics might be an additional way by which n-3 PUFAs exert their cellular effects. Concerning F-actin, this analysis did not reveal unmistakable results impeding a generalized conclusion.