RESUMO
Cell chemotaxis, such as migration of fibroblasts towards growth factors during development and wound healing, requires precise spatial coordination of signalling events. Phosphoinositides and signalling enzymes involved in their generation and hydrolysis have been implicated in regulation of chemotaxis; however, the role and importance of specific components remain poorly understood. Here, we demonstrate that phospholipase C epsilon (PLCε) contributes to fibroblast chemotaxis towards platelet-derived growth factor (PDGF-BB). Using PLCe1 null fibroblasts we show that cells deficient in PLCε have greatly reduced directionality towards PDGF-BB without detrimental effect on their basal ability to migrate. Furthermore, we show that in intact fibroblasts, signalling events, such as activation of Rac, are spatially compromised by the absence of PLCε that affects the ability of cells to enlarge their protrusions in the direction of the chemoattractant. By further application of live cell imaging and the use of FRET-based biosensors, we show that generation of Ins(1,4,5)P(3) and recruitment of PLCε are most pronounced in protrusions responding to the PDGF-BB gradient. Furthermore, the phospholipase C activity of PLCε is critical for its role in chemotaxis, consistent with the importance of Ins(1,4,5)P(3) generation and sustained calcium responses in this process. As PLCε has extensive signalling connectivity, using transgenic fibroblasts we ruled out its activation by direct binding to Ras or Rap GTPases, and suggest instead new unexpected links for PLCε in the context of chemotaxis.
Assuntos
Quimiotaxia/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fosfoinositídeo Fosfolipase C/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Animais , Células Cultivadas , Quimiotaxia/genética , Fibroblastos/citologia , Camundongos , Camundongos Transgênicos , Fosfoinositídeo Fosfolipase C/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genéticaRESUMO
Inhibition of ERK-MAPK signaling by expression of dominant-negative MEK1 in the tumor vasculature suppresses angiogenesis and tumor growth. In an organotypic tissue culture angiogenesis assay, ERK-MAPK inhibition during the migratory phase results in loss of bipolarity, detachment, and cell death of isolated endothelial cells and retraction of sprouting tubules. These effects are the consequence of upregulated Rho-kinase signaling. Transient inhibition of Rho-kinase rescues the effects of ERK-MAPK inhibition in vitro and in vivo, promotes sprouting, and increases vessel length in tumors. We propose a regulatory role of Rho-kinase by ERK-MAPK during angiogenesis that acts through the control of actomyosin contractility. Our data delineate a mechanism by which ERK-MAPK promotes endothelial cell survival and sprouting by downregulating Rho-kinase signaling.
Assuntos
Células Endoteliais/fisiologia , Endotélio Vascular/patologia , MAP Quinase Quinase 1/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Neovascularização Patológica/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Actomiosina/metabolismo , Animais , Movimento Celular , Polaridade Celular , Sobrevivência Celular , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/citologia , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , MAP Quinase Quinase 1/genética , Masculino , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Neovascularização Patológica/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Transdução de Sinais , Veias Umbilicais/citologia , Quinases Associadas a rhoRESUMO
Rho-associated kinases 1 and 2 (ROCK1/2) are Rho-GTPase effectors that control key aspects of the actin cytoskeleton, but their role in proliferation and cancer initiation or progression is not known. Here, we provide evidence that ROCK1 and ROCK2 act redundantly to maintain actomyosin contractility and cell proliferation and that their loss leads to cell-cycle arrest and cellular senescence. This phenotype arises from down-regulation of the essential cell-cycle proteins CyclinA, CKS1 and CDK1. Accordingly, while the loss of either Rock1 or Rock2 had no negative impact on tumorigenesis in mouse models of non-small cell lung cancer and melanoma, loss of both blocked tumor formation, as no tumors arise in which both Rock1 and Rock2 have been genetically deleted. Our results reveal an indispensable role for ROCK, yet redundant role for isoforms 1 and 2, in cell cycle progression and tumorigenesis, possibly through the maintenance of cellular contractility.
Assuntos
Carcinogênese , Proliferação de Células , Quinases Associadas a rho/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Técnicas de Inativação de Genes , Melanoma/patologia , Camundongos , Quinases Associadas a rho/genéticaRESUMO
There is an urgent need to identify new therapeutic opportunities for metastatic melanoma. Fragment-based screening has led to the discovery of orally available, ATP-competitive AKT kinase inhibitors, AT13148 and CCT129254. These compounds also inhibit the Rho-kinases ROCK 1 and ROCK 2 and we show they potently inhibit ROCK activity in melanoma cells in culture and in vivo. Treatment of melanoma cells with CCT129254 or AT13148 dramatically reduces cell invasion, impairing both "amoeboid-like" and mesenchymal-like modes of invasion in culture. Intravital imaging shows that CCT129254 or AT13148 treatment reduces the motility of melanoma cells in vivo. CCT129254 inhibits melanoma metastasis when administered 2 days after orthotopic intradermal injection of the cells, or when treatment starts after metastases have arisen. Mechanistically, our data suggest that inhibition of ROCK reduces the ability of melanoma cells to efficiently colonize the lungs. These results suggest that these novel inhibitors of ROCK may be beneficial in the treatment of metastasis.
Assuntos
2-Hidroxifenetilamina/análogos & derivados , Movimento Celular/efeitos dos fármacos , Melanoma/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Pirazóis/administração & dosagem , Quinases Associadas a rho/genética , 2-Hidroxifenetilamina/administração & dosagem , Linhagem Celular Tumoral , Humanos , Melanoma/genética , Melanoma/patologia , Invasividade Neoplásica/genética , Metástase Neoplásica , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
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/metabolismoRESUMO
RAL small GTPases, encoded by the Rala and Ralb genes, are members of the RAS superfamily of small GTPases and can act as downstream effectors of RAS [1]. Although highly similar, distinct functions have been identified for RALA and RALB: RALA has been implicated in epithelial cell polarity [2], insulin secretion [3], GLUT4 translocation [4, 5], neurite branching, and neuronal polarity [6, 7], and RALB in tumor cell survival [8], migration/invasion [9-12], TBK1 activation [13], and autophagy [14]. To investigate RAL GTPases in vivo, we generated null and conditional knockout mice. Ralb null mice are viable with no overt phenotype; the Rala null leads to exencephaly and embryonic lethality. The exencephaly phenotype is exacerbated in Rala(-/-);Ralb(+/-) embryos; embryos null for Rala and Ralb do not live past gastrulation. Using a Kras-driven non-small cell lung carcinoma mouse model, we found that either RALA or RALB is sufficient for tumor growth. However, deletion of both Ral genes blocks tumor formation. Either RALA or RALB is sufficient for cell proliferation, but cells lacking both fail to proliferate. These studies demonstrate functions of RAL proteins in development, tumorigenesis, and cell proliferation and show that RALA and RALB act in a redundant fashion.
Assuntos
Transformação Celular Neoplásica , Desenvolvimento Embrionário , Proteínas ral de Ligação ao GTP/fisiologia , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Polaridade Celular , Proliferação de Células , Células Cultivadas , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tubo Neural/embriologia , Tubo Neural/metabolismo , Defeitos do Tubo Neural/genética , Proteína Oncogênica p21(ras)/metabolismo , Transdução de Sinais , Proteínas ral de Ligação ao GTP/genéticaRESUMO
Proinflammatory cytokines are frequently observed in the tumor microenvironment, and chronic inflammation is involved in cancer initiation and progression. We show that cytokine signaling through the receptor subunit GP130-IL6ST and the kinase JAK1 generates actomyosin contractility through Rho-kinase dependent signaling. This pathway generates contractile force in stromal fibroblasts to remodel the extracellular matrix to create tracks for collective migration of squamous carcinoma cells and provides the high levels of actomyosin contractility required for migration of individual melanoma cells in the rounded, "amoeboid" mode. Thus, cytokine signaling can generate actomyosin contractility in both stroma and tumor cells. Strikingly, actomyosin contractility itself positively modulates activity of the transcription factor STAT3 downstream of JAK1, demonstrating positive feedback within the signaling network.
Assuntos
Actomiosina/metabolismo , Janus Quinase 1/metabolismo , Neoplasias/metabolismo , Transdução de Sinais , Células Estromais/metabolismo , Quinases Associadas a rho/metabolismo , Movimento Celular , Humanos , Melanoma/metabolismo , Melanoma/patologia , Neoplasias/patologia , Fator de Transcrição STAT3/metabolismo , Células Estromais/patologiaRESUMO
Glycogen synthase kinase 3beta (GSK3beta) is a serine/threonine kinase involved in insulin, growth factor and Wnt signalling. In Wnt signalling, GSK3beta is recruited to a multiprotein complex via interaction with axin, where it hyperphosphorylates beta-catenin, marking it for ubiquitylation and destruction. We have now determined the crystal structure of GSK3beta in complex with a minimal GSK3beta-binding segment of axin, at 2.4 A resolution. The structure confirms the co-localization of the binding sites for axin and FRAT in the C-terminal domain of GSK3beta, but reveals significant differences in the interactions made by axin and FRAT, mediated by conformational plasticity of the 285-299 loop in GSK3beta. Detailed comparison of the axin and FRAT GSK3beta complexes allows the generation of highly specific mutations, which abrogate binding of one or the other. Quantitative analysis suggests that the interaction of GSK3beta with the axin scaffold enhances phosphorylation of beta-catenin by >20 000-fold.