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[This corrects the article DOI: 10.1371/journal.pbio.1002125.].
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Patterning of functional blood vessel networks is achieved by pruning of superfluous connections. The cellular and molecular principles of vessel regression are poorly understood. Here we show that regression is mediated by dynamic and polarized migration of endothelial cells, representing anastomosis in reverse. Establishing and analyzing the first axial polarity map of all endothelial cells in a remodeling vascular network, we propose that balanced movement of cells maintains the primitive plexus under low shear conditions in a metastable dynamic state. We predict that flow-induced polarized migration of endothelial cells breaks symmetry and leads to stabilization of high flow/shear segments and regression of adjacent low flow/shear segments.
Assuntos
Vasos Sanguíneos/citologia , Endotélio Vascular/citologia , Animais , Polaridade Celular , Modelos BiológicosRESUMO
The hepatic hormone hepcidin is a key regulator of systemic iron metabolism. Its expression is largely regulated by 2 signaling pathways: the "iron-regulated" bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways. To obtain broader insights into cellular processes that modulate hepcidin transcription and to provide a resource to identify novel genetic modifiers of systemic iron homeostasis, we designed an RNA interference (RNAi) screen that monitors hepcidin promoter activity after the knockdown of 19 599 genes in hepatocarcinoma cells. Interestingly, many of the putative hepcidin activators play roles in signal transduction, inflammation, or transcription, and affect hepcidin transcription through BMP-responsive elements. Furthermore, our work sheds light on new components of the transcriptional machinery that maintain steady-state levels of hepcidin expression and its responses to the BMP- and interleukin-6-triggered signals. Notably, we discover hepcidin suppression mediated via components of Ras/RAF MAPK and mTOR signaling, linking hepcidin transcriptional control to the pathways that respond to mitogen stimulation and nutrient status. Thus using a combination of RNAi screening, reverse phase protein arrays, and small molecules testing, we identify links between the control of systemic iron homeostasis and critical liver processes such as regeneration, response to injury, carcinogenesis, and nutrient metabolism.
Assuntos
Regulação da Expressão Gênica , Hepcidinas/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Interferência de RNA , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular , Perfilação da Expressão Gênica , Hepcidinas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Ligação Proteica , Reprodutibilidade dos Testes , Elementos de Resposta , Transcrição GênicaRESUMO
Immune signalling pathways need to be tightly regulated as overactivation of these pathways can result in chronic inflammatory diseases and cancer. NF-κB signalling and associated innate immune pathways are crucial in the first line of defense against infection in all animals. In a genome-wide RNAi screen for modulators of Drosophila immune deficiency (IMD)/NF-κB signalling, we identified components of the Ras/MAPK pathway as essential for suppression of IMD pathway activity, even in the absence of an immune challenge. Downregulation of Ras/MAPK activity mimics the induction of innate immune responses by microbial patterns. Conversely, ectopic Ras/MAPK pathway activation results in the suppression of Drosophila IMD/NF-κB signalling. Mechanistically, we show that the Ras/MAPK pathway acts by inducing transcription of the IMD pathway inhibitor Pirk/Rudra/PIMS. Finally, in vivo experiments demonstrate a requirement for Ras/MAPK signalling in restricting innate immune responses in haemocytes, fat body and adult intestinal stem cells. Our observations provide an example of a pathway that promotes cell proliferation and has simultaneously been utilized to limit the immune response.
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Proteínas de Drosophila/biossíntese , Drosophila melanogaster/imunologia , Imunidade Inata , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteínas ras/metabolismo , Animais , Bactérias/imunologia , Proteínas de Drosophila/metabolismo , Corpo Adiposo/imunologia , Regulação da Expressão Gênica , Hemócitos/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Células-Tronco/imunologiaRESUMO
Hepcidin is the master regulatory hormone of systemic iron metabolism. Hepcidin deficiency causes common iron overload syndromes whereas its overexpression is responsible for microcytic anemias. Hepcidin transcription is activated by the bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways, whereas comparatively little is known about how hepcidin expression is inhibited. By using high-throughput siRNA screening we identified SMAD7 as a potent hepcidin suppressor. SMAD7 is an inhibitory SMAD protein that mediates a negative feedback loop to both transforming growth factor-beta and BMP signaling and that recently was shown to be coregulated with hepcidin via SMAD4 in response to altered iron availability in vivo. We show that SMAD7 is coregulated with hepcidin by BMPs in primary murine hepatocytes and that SMAD7 overexpression completely abolishes hepcidin activation by BMPs and transforming growth factor-beta. We identify a distinct SMAD regulatory motif (GTCAAGAC) within the hepcidin promoter involved in SMAD7-dependent hepcidin suppression, demonstrating that SMAD7 does not simply antagonize the previously reported hemojuvelin/BMP-responsive elements. This work identifies a potent inhibitory factor for hepcidin expression and uncovers a negative feedback pathway for hepcidin regulation, providing insight into a mechanism how hepcidin expression may be limited to avoid iron deficiency.
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Peptídeos Catiônicos Antimicrobianos/biossíntese , Hepatócitos/metabolismo , Ferro/metabolismo , Proteína Smad7/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Proteínas Morfogenéticas Ósseas/farmacologia , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Regulação para Baixo , Retroalimentação Fisiológica , Hepatócitos/efeitos dos fármacos , Hepcidinas , Humanos , Interleucina-6/farmacologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteína Smad7/antagonistas & inibidores , Proteína Smad7/química , Proteína Smad7/genética , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Many pleiotropic roles have been ascribed to small abundant HMG-Box (HMGB) proteins in higher eukaryotes but their precise function has remained enigmatic. To investigate their function genetically we have generated a defined deficiency uncovering the functionally redundant genes encoding HMGD and HMGZ, the Drosophila counterparts of HMGB1-3 in mammals. The resulting mutant is a strong hypomorphic allele of HmgD/Z. Surprisingly this allele is viable and exhibits only minor morphological defects even when homozygous. However, this allele interacts strongly with mutants of the Brahma chromatin remodeling complex, while no interaction was observed with mutant alleles of other remodeling complexes. We also observe genetic interactions between the HmgD/Z deficiency and some, but not all, known Brahma targets. These include the homeotic genes Sex combs reduced and Antennapedia, as well as the gene encoding the cell-signaling protein Rhomboid. In contrast to more general structural roles previously suggested for these proteins, we infer that a major function of the abundant HMGB proteins in Drosophila is to participate in Brahma-dependent chromatin remodeling at a specific subset of Brahma-dependent promoters.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Montagem e Desmontagem da Cromatina/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Transativadores/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas de Grupo de Alta Mobilidade/deficiência , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Regiões Promotoras Genéticas , Transativadores/genética , Asas de Animais/anatomia & histologiaRESUMO
Animal cell shape is largely determined by the cortex, a thin actin network underlying the plasma membrane in which myosin-driven stresses generate contractile tension. Tension gradients result in local contractions and drive cell deformations. Previous cortical tension regulation studies have focused on myosin motors. Here, we show that cortical actin network architecture is equally important. First, we observe that actin cortex thickness and tension are inversely correlated during cell-cycle progression. We then show that the actin filament length regulators CFL1, CAPZB and DIAPH1 regulate mitotic cortex thickness and find that both increasing and decreasing thickness decreases tension in mitosis. This suggests that the mitotic cortex is poised close to a tension maximum. Finally, using a computational model, we identify a physical mechanism by which maximum tension is achieved at intermediate actin filament lengths. Our results indicate that actin network architecture, alongside myosin activity, is key to cell surface tension regulation.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Ciclo Celular , Forma Celular , Mecanotransdução Celular , Citoesqueleto de Actina/ultraestrutura , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína de Capeamento de Actina CapZ/genética , Proteína de Capeamento de Actina CapZ/metabolismo , Cofilina 1/genética , Cofilina 1/metabolismo , Simulação por Computador , Forminas , Células HeLa , Humanos , Interfase , Mitose , Modelos Biológicos , Tensão Superficial , TransfecçãoRESUMO
There is evidence that HMGB proteins facilitate, while linker histones inhibit chromatin remodelling, respectively. We have examined the effects of HMG-D and histone H1/H5 on accessibility of nucleosomal DNA. Using the 601.2 nucleosome positioning sequence designed by Widom and colleagues we assembled nucleosomes in vitro and probed DNA accessibility with restriction enzymes in the presence or absence of HMG-D and histone H1/H5. For HMG-D our results show increased digestion at two spatially adjacent sites, the dyad and one terminus of nucleosomal DNA. Elsewhere varying degrees of protection from digestion were observed. The C-terminal acidic tail of HMG-D is essential for this pattern of accessibility. Neither the HMG domain by itself nor in combination with the adjacent basic region is sufficient. Histone H1/H5 binding produces two sites of increased digestion on opposite faces of the nucleosome and decreased digestion at all other sites. Our results provide the first evidence of local changes in the accessibility of nucleosomal DNA upon separate interaction with two linker binding proteins.
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DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histonas/metabolismo , Nucleossomos/metabolismo , Animais , Galinhas , DNA/química , DNA/genética , Enzimas de Restrição do DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/genética , Mutação/genética , Ensaios de Proteção de Nucleases , Nucleossomos/química , Nucleossomos/genética , Ligação ProteicaRESUMO
Formation of a regularly branched blood vessel network is crucial in development and physiology. Here we show that the expression of the Notch ligand Dll4 fluctuates in individual endothelial cells within sprouting vessels in the mouse retina in vivo and in correlation with dynamic cell movement in mouse embryonic stem cell-derived sprouting assays. We also find that sprout elongation and branching associates with a highly differential phase pattern of Dll4 between endothelial cells. Stimulation with pathologically high levels of Vegf, or overexpression of Dll4, leads to Notch dependent synchronization of Dll4 fluctuations within clusters, both in vitro and in vivo. Our results demonstrate that the Vegf-Dll4/Notch feedback system normally operates to generate heterogeneity between endothelial cells driving branching, whilst synchronization drives vessel expansion. We propose that this sensitive phase transition in the behaviour of the Vegf-Dll4/Notch feedback loop underlies the morphogen function of Vegfa in vascular patterning.
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Neoplasias Encefálicas/genética , Células Endoteliais/metabolismo , Glioblastoma/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Neovascularização Patológica/genética , Receptores Notch/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proteínas de Ligação ao Cálcio , Movimento Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Retroalimentação Fisiológica , Regulação da Expressão Gênica , Genes Reporter , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/efeitos dos fármacos , Células-Tronco Embrionárias Murinas/metabolismo , Transplante de Neoplasias , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neovascularização Fisiológica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Notch/genética , Retina/citologia , Retina/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Endothelial cells respond to molecular and physical forces in development and vascular homeostasis. Deregulation of endothelial responses to flow-induced shear is believed to contribute to many aspects of cardiovascular diseases including atherosclerosis. However, how molecular signals and shear-mediated physical forces integrate to regulate vascular patterning is poorly understood. Here we show that endothelial non-canonical Wnt signalling regulates endothelial sensitivity to shear forces. Loss of Wnt5a/Wnt11 renders endothelial cells more sensitive to shear, resulting in axial polarization and migration against flow at lower shear levels. Integration of flow modelling and polarity analysis in entire vascular networks demonstrates that polarization against flow is achieved differentially in artery, vein, capillaries and the primitive sprouting front. Collectively our data suggest that non-canonical Wnt signalling stabilizes forming vascular networks by reducing endothelial shear sensitivity, thus keeping vessels open under low flow conditions that prevail in the primitive plexus.
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Células Endoteliais/fisiologia , Estresse Mecânico , Remodelação Vascular , Via de Sinalização Wnt , Animais , Linhagem Celular , Movimento Celular , Polaridade Celular , Regulação da Expressão Gênica , CamundongosRESUMO
Sprouting angiogenesis drives blood vessel growth in healthy and diseased tissues. Vegf and Dll4/Notch signalling cooperate in a negative feedback loop that specifies endothelial tip and stalk cells to ensure adequate vessel branching and function. Current concepts posit that endothelial cells default to the tip-cell phenotype when Notch is inactive. Here we identify instead that the stalk-cell phenotype needs to be actively repressed to allow tip-cell formation. We show this is a key endothelial function of neuropilin-1 (Nrp1), which suppresses the stalk-cell phenotype by limiting Smad2/3 activation through Alk1 and Alk5. Notch downregulates Nrp1, thus relieving the inhibition of Alk1 and Alk5, thereby driving stalk-cell behaviour. Conceptually, our work shows that the heterogeneity between neighbouring endothelial cells established by the lateral feedback loop of Dll4/Notch utilizes Nrp1 levels as the pivot, which in turn establishes differential responsiveness to TGF-ß/BMP signalling.
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Receptores de Ativinas Tipo I/metabolismo , Endotélio Vascular/crescimento & desenvolvimento , Neuropilina-1/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Notch/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Receptores de Activinas Tipo II , Animais , Fator 2 de Diferenciação de Crescimento/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Fenótipo , Receptor do Fator de Crescimento Transformador beta Tipo I , Proteína Smad2/metabolismo , Proteína Smad3/metabolismoRESUMO
Coordinated activity of VEGF and Notch signals guides the endothelial cell (EC) specification into tip and stalk cells during angiogenesis. Notch activation in stalk cells leads to proliferation arrest via an unknown mechanism. By using gain- and loss-of-function gene-targeting approaches, here we show that PTEN is crucial for blocking stalk cell proliferation downstream of Notch, and this is critical for mouse vessel development. Endothelial deletion of PTEN results in vascular hyperplasia due to a failure to mediate Notch-induced proliferation arrest. Conversely, overexpression of PTEN reduces vascular density and abrogates the increase in EC proliferation induced by Notch blockade. PTEN is a lipid/protein phosphatase that also has nuclear phosphatase-independent functions. We show that both the catalytic and non-catalytic APC/C-Fzr1/Cdh1-mediated activities of PTEN are required for stalk cells' proliferative arrest. These findings define a Notch-PTEN signalling axis as an orchestrator of vessel density and implicate the PTEN-APC/C-Fzr1/Cdh1 hub in angiogenesis.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdh1/metabolismo , Proliferação de Células/genética , Células Endoteliais/metabolismo , Neovascularização Fisiológica/genética , PTEN Fosfo-Hidrolase/genética , RNA Mensageiro/metabolismo , Receptores Notch/metabolismo , Animais , Imunofluorescência , Immunoblotting , Camundongos , PTEN Fosfo-Hidrolase/metabolismo , Reação em Cadeia da PolimeraseRESUMO
Innate immune signalling pathways are evolutionarily conserved between invertebrates and vertebrates. The analysis of NF-kappaB signalling in Drosophila has contributed important insights into how organisms respond to infection. Nevertheless, significant gaps remain in our understanding of how the activation of intracellular signalling elicits specific transcriptional programs. Here we report a genome-wide RNA interference survey for transcription factors that are required for Toll-dependent immune responses. In addition to the NF-kappaB homologs Dif, Dorsal and factors of the general transcription machinery, we identified Deformed Epidermal Autoregulatory Factor 1 (Deaf1) to be required for the expression of the Toll target gene Drosomycin in cultured cells and in Drosophila in vivo. We show that Deaf1 is required for the survival of flies after fungal, but not E. coli, infection. We determine that Deaf1 acts downstream of the NF-kappaB factors Dorsal and Dif. These results indicate that Deaf1 is an important contributor to innate immune responses in vivo.
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Proteínas de Drosophila/metabolismo , Drosophila/imunologia , Regulação da Expressão Gênica , Imunidade Inata , Proteínas Nucleares/metabolismo , Interferência de RNA , Animais , Células Cultivadas , Proteínas de Ligação a DNA , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Genes de Insetos , Genômica , Proteínas Nucleares/química , Proteínas Nucleares/genética , Transdução de Sinais , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Despite extensive study of heterochromatin, relatively little is known about the mechanisms by which such a structure forms. We show that the Drosophila homologue of the human alpha-thalassemia and mental retardation X-linked protein (dATRX), is important in the formation or maintenance of heterochromatin through modification of position effect variegation. We further show that there are two isoforms of the dATRX protein, the longer of which interacts directly with heterochromatin protein 1 (dHP-1) through a CxVxL motif both in vitro and in vivo. These two proteins co-localise at heterochromatin in a manner dependent on this motif. Consistent with this observation, the long isoform of the dATRX protein localises primarily to the heterochromatin at the chromocentre on salivary gland polytene chromosomes, whereas the short isoform binds to many sites along the chromosome arms. We suggest that the establishment of a regular nucleosomal organisation may be common to heterochromatin and transcriptionally repressed chromatin in other locations, and may require the action of ATP dependent chromatin remodelling factors.