Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 6 de 6
Filtrar
Mais filtros

Base de dados
Tipo de documento
País de afiliação
Intervalo de ano de publicação
1.
J Cell Sci ; 132(19)2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31515278

RESUMO

Tumor stem cells and malignant multicellular networks have been separately implicated in the therapeutic resistance of glioblastoma multiforme (GBM), the most aggressive type of brain cancer in adults. Here, we show that small-molecule inhibition of RHO-associated serine/threonine kinase proteins (ROCKi) significantly promoted the outgrowth of neurite-like cell projections in cultures of heterogeneous patient-derived GBM stem-like cells. These projections formed de novo-induced cellular network (iNet) 'webs', which regressed after withdrawal of ROCKi. Connected cells within the iNet web exhibited long range Ca2+ signal transmission, and significant lysosomal and mitochondrial trafficking. In contrast to their less-connected vehicle control counterparts, iNet cells remained viable and proliferative after high-dose radiation. These findings demonstrate a link between ROCKi-regulated cell projection dynamics and the formation of radiation-resistant multicellular networks. Our study identifies means to reversibly induce iNet webs ex vivo, and may thereby accelerate future studies into the biology of GBM cellular networks.


Assuntos
Glioblastoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neuritos/metabolismo , Sinalização do Cálcio/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Humanos , Immunoblotting , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Crescimento Neuronal/fisiologia , Fenótipo , Proteínas Serina-Treonina Quinases/metabolismo
2.
Ann Rheum Dis ; 77(3): 431-440, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29259049

RESUMO

OBJECTIVES: Systemic sclerosis (SSc) is characterised by tissue fibrosis and vasculopathy with defective angiogenesis. Transforming growth factor beta (TGF-ß) plays a major role in tissue fibrosis, including downregulation of caveolin-1 (Cav-1); however, its role in defective angiogenesis is less clear. Pigment epithelium-derived factor (PEDF), a major antiangiogenic factor, is abundantly secreted by SSc fibroblasts. Here, we investigated the effect of TGF-ß and Cav-1 on PEDF expression and the role of PEDF in the ability of SSc fibroblasts to modulate angiogenesis. METHODS: PEDF and Cav-1 expression in fibroblasts and endothelial cells were evaluated by means of immunohistochemistry on human and mouse skin biopsies. PEDF and Cav-1 were silenced in cultured SSc and control fibroblasts using lentiviral short-hairpin RNAs. Organotypic fibroblast-endothelial cell co-cultures and matrigel assays were employed to assess angiogenesis. RESULTS: PEDF is highly expressed in myofibroblasts and reticular fibroblasts with low Cav-1 expression in SSc skin biopsies, and it is induced by TGF-ß in vitro. SSc fibroblasts suppress angiogenesis in an organotypic model. This model is reproduced by silencing Cav-1 in normal dermal fibroblasts. Conversely, silencing PEDF in SSc fibroblasts rescues their antiangiogenic phenotype. Consistently, transgenic mice with TGF-ß receptor hyperactivation show lower Cav-1 and higher PEDF expression levels in skin biopsies accompanied by reduced blood vessel density. CONCLUSIONS: Our data reveal a new pathway by which TGF-ß suppresses angiogenesis in SSc, through decreased fibroblast Cav-1 expression and subsequent PEDF secretion. This pathway may present a promising target for new therapeutic interventions in SSc.


Assuntos
Caveolina 1/metabolismo , Proteínas do Olho/metabolismo , Fibroblastos/metabolismo , Neovascularização Patológica/metabolismo , Fatores de Crescimento Neural/metabolismo , Escleroderma Sistêmico/patologia , Serpinas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Células Cultivadas , Células Endoteliais/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Escleroderma Sistêmico/metabolismo , Pele/patologia
3.
Arterioscler Thromb Vasc Biol ; 31(3): 657-64, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21148427

RESUMO

OBJECTIVE: RhoJ/TCL was identified by our group as an endothelial-expressed Rho GTPase. The aim of this study was to determine its tissue distribution, subcellular localization, and function in endothelial migration and tube formation. METHODS AND RESULTS: Using in situ hybridization, RhoJ was localized to endothelial cells in a set of normal and cancerous tissues and in the vasculature of mouse embryos; endogenous RhoJ was localized to focal adhesions by immunofluorescence. The proangiogenic factor vascular endothelial growth factor activated RhoJ in endothelial cells. Using either small interfering (si)RNA-mediated knockdown of RhoJ expression or overexpression of constitutively active RhoJ (daRhoJ), RhoJ was found to positively regulate endothelial motility and tubule formation. Downregulating RhoJ expression increased focal adhesions and stress fibers in migrating cells, whereas daRhoJ overexpression resulted in the converse. RhoJ downregulation resulted in increased contraction of a collagen gel and increased phospho-myosin light chain, indicative of increased actomyosin contractility. Pharmacological inhibition of Rho-kinase (which phosphorylates myosin light chain) or nonmuscle myosin II reversed the defective tube formation and migration of RhoJ knockdown cells. CONCLUSIONS: RhoJ is endothelial-expressed in vivo, activated by vascular endothelial growth factor, localizes to focal adhesions, regulates endothelial cell migration and tube formation, and modulates actomyosin contractility and focal adhesion numbers.


Assuntos
Actomiosina/metabolismo , Movimento Celular , Células Endoteliais/enzimologia , Adesões Focais/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Neovascularização Fisiológica , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Movimento Celular/efeitos dos fármacos , Forma Celular , Células Cultivadas , Células Endoteliais/efeitos dos fármacos , Imunofluorescência , GTP Fosfo-Hidrolases/genética , Humanos , Hibridização In Situ , Camundongos , Cadeias Leves de Miosina/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Fibras de Estresse/metabolismo , Transfecção , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas rho de Ligação ao GTP/genética , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismo
4.
Cell Rep ; 12(10): 1584-93, 2015 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-26321638

RESUMO

Within a 3D tissue, cells need to integrate signals from growth factors, such as BMPs, and the extracellular matrix (ECM) to coordinate growth and differentiation. Here, we use the Drosophila embryo as a model to investigate how BMP responses are influenced by a cell's local ECM environment. We show that integrins, which are ECM receptors, are absolutely required for peak BMP signaling. This stimulatory effect of integrins requires their intracellular signaling function, which is activated by the ECM protein collagen IV. Mechanistically, integrins interact with the BMP receptor and stimulate phosphorylation of the downstream Mad transcription factor. The BMP-pathway-enhancing function of integrins is independent of focal adhesion kinase, but it requires conserved NPXY motifs in the ß-integrin cytoplasmic tail. Furthermore, we show that an α-integrin subunit is a BMP target gene, identifying positive feedback between integrin signaling and BMP pathway activity that may contribute to robust cell fate decisions.


Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Integrinas/fisiologia , Animais , Linhagem Celular , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Drosophila melanogaster/embriologia , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário , Retroalimentação Fisiológica , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Transdução de Sinais
5.
Nat Commun ; 6: 7286, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-26129894

RESUMO

During angiogenesis, Rho-GTPases influence endothelial cell migration and cell-cell adhesion; however it is not known whether they control formation of vessel lumens, which are essential for blood flow. Here, using an organotypic system that recapitulates distinct stages of VEGF-dependent angiogenesis, we show that lumen formation requires early cytoskeletal remodelling and lateral cell-cell contacts, mediated through the RAC1 guanine nucleotide exchange factor (GEF) DOCK4 (dedicator of cytokinesis 4). DOCK4 signalling is necessary for lateral filopodial protrusions and tubule remodelling prior to lumen formation, whereas proximal, tip filopodia persist in the absence of DOCK4. VEGF-dependent Rac activation via DOCK4 is necessary for CDC42 activation to signal filopodia formation and depends on the activation of RHOG through the RHOG GEF, SGEF. VEGF promotes interaction of DOCK4 with the CDC42 GEF DOCK9. These studies identify a novel Rho-family GTPase activation cascade for the formation of endothelial cell filopodial protrusions necessary for tubule remodelling, thereby influencing subsequent stages of lumen morphogenesis.


Assuntos
Proteínas Ativadoras de GTPase/fisiologia , Neovascularização Patológica , Neovascularização Fisiológica , Pseudópodes/fisiologia , Animais , Citoesqueleto/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA