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1.
Biotechnol Biofuels Bioprod ; 16(1): 137, 2023 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-37710260

RESUMO

Clostridium thermocellum is a natively cellulolytic bacterium that is promising candidate for cellulosic biofuel production, and can produce ethanol at high yields (75-80% of theoretical) but the ethanol titers produced thus far are too low for commercial application. In several strains of C. thermocellum engineered for increased ethanol yield, ethanol titer seems to be limited by ethanol tolerance. Previous work to improve ethanol tolerance has focused on the WT organism. In this work, we focused on understanding ethanol tolerance in several engineered strains of C. thermocellum. We observed a tradeoff between ethanol tolerance and production. Adaptation for increased ethanol tolerance decreases ethanol production. Second, we observed a consistent genetic response to ethanol stress involving mutations at the AdhE locus. These mutations typically reduced NADH-linked ADH activity. About half of the ethanol tolerance phenotype could be attributed to the elimination of NADH-linked activity based on a targeted deletion of adhE. Finally, we observed that rich growth medium increases ethanol tolerance, but this effect is eliminated in an adhE deletion strain. Together, these suggest that ethanol inhibits growth and metabolism via a redox-imbalance mechanism. The improved understanding of mechanisms of ethanol tolerance described here lays a foundation for developing strains of C. thermocellum with improved ethanol production.

2.
Biotechnol Biofuels ; 13: 40, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32175007

RESUMO

BACKGROUND: Engineering efforts targeted at increasing ethanol by modifying the central fermentative metabolism of Clostridium thermocellum have been variably successful. Here, we aim to understand this variation by a multifaceted approach including genomic and transcriptomic analysis combined with chemostat cultivation and high solids cellulose fermentation. Three strain lineages comprising 16 strains total were examined. Two strain lineages in which genes involved in pathways leading to organic acids and/or sporulation had been knocked out resulted in four end-strains after adaptive laboratory evolution (ALE). A third strain lineage recapitulated mutations involving adhE that occurred spontaneously in some of the engineered strains. RESULTS: Contrary to lactate dehydrogenase, deleting phosphotransacetylase (pta, acetate) negatively affected steady-state biomass concentration and caused increased extracellular levels of free amino acids and pyruvate, while no increase in ethanol was detected. Adaptive laboratory evolution (ALE) improved growth and shifted elevated levels of amino acids and pyruvate towards ethanol, but not for all strain lineages. Three out of four end-strains produced ethanol at higher yield, and one did not. The occurrence of a mutation in the adhE gene, expanding its nicotinamide-cofactor compatibility, enabled two end-strains to produce more ethanol. A disruption in the hfsB hydrogenase is likely the reason why a third end-strain was able to make more ethanol. RNAseq analysis showed that the distribution of fermentation products was generally not regulated at the transcript level. At 120 g/L cellulose loadings, deletions of spo0A, ldh and pta and adaptive evolution did not negatively influence cellulose solubilization and utilization capabilities. Strains with a disruption in hfsB or a mutation in adhE produced more ethanol, isobutanol and 2,3-butanediol under these conditions and the highest isobutanol and ethanol titers reached were 5.1 and 29.9 g/L, respectively. CONCLUSIONS: Modifications in the organic acid fermentative pathways in Clostridium thermocellum caused an increase in extracellular pyruvate and free amino acids. Adaptive laboratory evolution led to improved growth, and an increase in ethanol yield and production due a mutation in adhE or a disruption in hfsB. Strains with deletions in ldh and pta pathways and subjected to ALE demonstrated undiminished cellulolytic capabilities when cultured on high cellulose loadings.

3.
Metab Eng Commun ; 10: e00116, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31890588

RESUMO

The robust lignocellulose-solubilizing activity of C. thermocellum makes it a top candidate for consolidated bioprocessing for biofuel production. Genetic techniques for C. thermocellum have lagged behind model organisms thus limiting attempts to improve biofuel production. To improve our ability to engineer C. thermocellum, we characterized a native Type I-B and heterologous Type II Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)/cas (CRISPR associated) systems. We repurposed the native Type I-B system for genome editing. We tested three thermophilic Cas9 variants (Type II) and found that GeoCas9, isolated from Geobacillus stearothermophilus, is active in C. thermocellum. We employed CRISPR-mediated homology directed repair to introduce a nonsense mutation into pyrF. For both editing systems, homologous recombination between the repair template and the genome appeared to be the limiting step. To overcome this limitation, we tested three novel thermophilic recombinases and demonstrated that exo/beta homologs, isolated from Acidithiobacillus caldus, are functional in C. thermocellum. For the Type I-B system an engineered strain, termed LL1586, yielded 40% genome editing efficiency at the pyrF locus and when recombineering machinery was expressed this increased to 71%. For the Type II GeoCas9 system, 12.5% genome editing efficiency was observed and when recombineering machinery was expressed, this increased to 94%. By combining the thermophilic CRISPR system (either Type I-B or Type II) with the recombinases, we developed a new tool that allows for efficient CRISPR editing. We are now poised to enable CRISPR technologies to better engineer C. thermocellum for both increased lignocellulose degradation and biofuel production.

4.
PLoS One ; 13(4): e0195143, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29621294

RESUMO

Thermoanaerobacterium saccharolyticum is a thermophilic anaerobe that has been engineered to produce high amounts of ethanol, reaching ~90% theoretical yield at a titer of 70 g/L. Here we report the physiological changes that occur upon deleting the redox-sensing transcriptional regulator Rex in wild type T. saccharolyticum: a single deletion of rex resulted in a two-fold increase in ethanol yield (from 40% to 91% theoretical yield), but the resulting strains grew only about a third as fast as the wild type strain. Deletion of the rex gene also had the effect of increasing expression of alcohol dehydrogenase genes, adhE and adhA. After several serial transfers, the ethanol yield decreased from an average of 91% to 55%, and the growth rates had increased. We performed whole-genome resequencing to identify secondary mutations in the Δrex strains adapted for faster growth. In several cases, secondary mutations had appeared in the adhE gene. Furthermore, in these strains the NADH-linked alcohol dehydrogenase activity was greatly reduced. Complementation studies were done to reintroduce rex into the Δrex strains: reintroducing rex decreased ethanol yield to below wild type levels in the Δrex strain without adhE mutations, but did not change the ethanol yield in the Δrex strain where an adhE mutation occurred.


Assuntos
Etanol/metabolismo , Produtos do Gene rex/genética , Produtos do Gene rex/metabolismo , Thermoanaerobacterium/genética , Thermoanaerobacterium/metabolismo , Adaptação Biológica , Álcool Desidrogenase/metabolismo , Fermentação , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Mutação , Oxirredução , Sequenciamento Completo do Genoma
5.
Biotechnol Biofuels ; 10: 276, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29213320

RESUMO

BACKGROUND: Clostridium thermocellum is a promising microorganism for conversion of cellulosic biomass to biofuel, without added enzymes; however, the low ethanol titer produced by strains developed thus far is an obstacle to industrial application. RESULTS: Here, we analyzed changes in the relative concentration of intracellular metabolites in response to gradual addition of ethanol to growing cultures. For C. thermocellum, we observed that ethanol tolerance, in experiments with gradual ethanol addition, was twofold higher than previously observed in response to a stepwise increase in the ethanol concentration, and appears to be due to a mechanism other than mutation. As ethanol concentrations increased, we found accumulation of metabolites upstream of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction and depletion of metabolites downstream of that reaction. This pattern was not observed in the more ethanol-tolerant organism Thermoanaerobacterium saccharolyticum. We hypothesize that the Gapdh enzyme may have different properties in the two organisms. Our hypothesis is supported by enzyme assays showing greater sensitivity of the C. thermocellum enzyme to high levels of NADH, and by the increase in ethanol tolerance and production when the T. saccharolyticum gapdh was expressed in C. thermocellum. CONCLUSIONS: We have demonstrated that a metabolic bottleneck occurs at the GAPDH reaction when the growth of C. thermocellum is inhibited by high levels of ethanol. We then showed that this bottleneck could be relieved by expression of the gapdh gene from T. saccharolyticum. This enzyme is a promising target for future metabolic engineering work.

6.
Metab Eng ; 42: 175-184, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28663138

RESUMO

Clostridium thermocellum ferments cellulose, is a promising candidate for ethanol production from cellulosic biomass, and has been the focus of studies aimed at improving ethanol yield. Thermoanaerobacterium saccharolyticum ferments hemicellulose, but not cellulose, and has been engineered to produce ethanol at high yield and titer. Recent research has led to the identification of four genes in T. saccharolyticum involved in ethanol production: adhE, nfnA, nfnB and adhA. We introduced these genes into C. thermocellum and observed significant improvements to ethanol yield, titer, and productivity. The four genes alone, however, were insufficient to achieve in C. thermocellum the ethanol yields and titers observed in engineered T. saccharolyticum strains, even when combined with gene deletions targeting hydrogen production. This suggests that other parts of T. saccharolyticum metabolism may also be necessary to reproduce the high ethanol yield and titer phenotype in C. thermocellum.


Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Clostridium thermocellum/metabolismo , Etanol/metabolismo , Thermoanaerobacterium/genética , Clostridium thermocellum/genética , Thermoanaerobacterium/enzimologia
7.
Metab Eng Commun ; 3: 120-129, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29142822

RESUMO

Clostridium thermocellum is a promising candidate for ethanol production from cellulosic biomass, but requires metabolic engineering to improve ethanol yield. A key gene in the ethanol production pathway is the bifunctional aldehyde and alcohol dehydrogenase, adhE. To explore the effects of overexpressing wild-type, mutant, and exogenous adhEs, we developed a new expression plasmid, pDGO144, that exhibited improved transformation efficiency and better gene expression than its predecessor, pDGO-66. This new expression plasmid will allow for many other metabolic engineering and basic research efforts in C. thermocellum. As proof of concept, we used this plasmid to express 12 different adhE genes (both wild type and mutant) from several organisms. Ethanol production varied between clones immediately after transformation, but tended to converge to a single value after several rounds of serial transfer. The previously described mutant C. thermocellum D494G adhE gave the best ethanol production, which is consistent with previously published results.

8.
Biotechnol Biofuels ; 8: 138, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26379770

RESUMO

BACKGROUND: Thermoanaerobacter saccharolyticum is a thermophilic microorganism that has been engineered to produce ethanol at high titer (30-70 g/L) and greater than 90 % theoretical yield. However, few genes involved in pyruvate to ethanol production pathway have been unambiguously identified. In T. saccharolyticum, the products of six putative pfor gene clusters and one pfl gene may be responsible for the conversion of pyruvate to acetyl-CoA. To gain insights into the physiological roles of PFOR and PFL, we studied the effect of deletions of several genes thought to encode these activities. RESULTS: It was found that pyruvate ferredoxin oxidoreductase enzyme (PFOR) is encoded by the pforA gene and plays a key role in pyruvate dissimilation. We further demonstrated that pyruvate formate-lyase activity (PFL) is encoded by the pfl gene. Although the pfl gene is normally expressed at low levels, it is crucial for biosynthesis in T. saccharolyticum. In pforA deletion strains, pfl expression increased and was able to partially compensate for the loss of PFOR activity. Deletion of both pforA and pfl resulted in a strain that required acetate and formate for growth and produced lactate as the primary fermentation product, achieving 88 % theoretical lactate yield. CONCLUSION: PFOR encoded by Tsac_0046 and PFL encoded by Tsac_0628 are only two routes for converting pyruvate to acetyl-CoA in T. saccharolyticum. The physiological role of PFOR is pyruvate dissimilation, whereas that of PFL is supplying C1 units for biosynthesis.

9.
Cytoskeleton (Hoboken) ; 72(8): 373-87, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26265212

RESUMO

In mice and humans, loss of myosin VI (Myo6) function results in deafness, and certain Myo6 mutations also result in cardiomyopathies in humans. The current studies have utilized the Snell's waltzer (sv) mouse (a functional null mutation for Myo6) to determine if this mouse also exhibits cardiac defects and thus used to determine the cellular and molecular basis for Myo6-associated heart disease. Myo6 is expressed in mouse heart where it is predominantly expressed in vascular endothelial cells (VECs) based on co-localization with the VEC cell marker CD31. Sv/sv heart mass is significantly greater than that of sv/+ littermates, a result of left ventricle hypertrophy. The left ventricle of the sv/sv exhibits extensive fibrosis, both interstitial and perivascular, based on histologic staining, and immunolocalization of several markers for fibrosis including fibronectin, collagen IV, and the fibroblast marker vimentin. Myo6 is also expressed in lung VECs but not in VECs of intestine, kidney, or liver. Sv/sv lungs exhibit increased periaveolar fibrosis and enlarged air sacs. Electron microscopy of sv/sv cardiac and lung VECs revealed abnormal ultrastructure, including luminal protrusions and increased numbers of cytoplasmic vesicles. Previous studies have shown that loss of function of either Myo6 or its adaptor binding partner synectin/GIPC results in impaired arterial development due to defects in VEGF signaling. However, examination of synectin/GIPC-/- heart revealed no fibrosis or significantly altered VEC ultrastructure, suggesting that the cardiac and lung defects observed in the sv/sv mouse are not due to Myo6 function in arterial development.


Assuntos
Cardiomiopatias/etiologia , Células Endoteliais , Hipertrofia Ventricular Esquerda , Cadeias Pesadas de Miosina/genética , Animais , Cardiomiopatias/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Fibrose , Humanos , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/patologia , Pulmão/patologia , Camundongos , Camundongos Mutantes , Miocárdio/patologia , Cadeias Pesadas de Miosina/metabolismo
10.
Metab Eng Commun ; 2: 23-29, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34150505

RESUMO

A key tool for metabolic engineering is the ability to express heterologous genes. One obstacle to gene expression in non-model organisms, and especially in relatively uncharacterized bacteria, is the lack of well-characterized promoters. Here we test 17 promoter regions for their ability to drive expression of the reporter genes ß-galactosidase (lacZ) and NADPH-alcohol dehydrogenase (adhB) in Clostridium thermocellum, an important bacterium for the production of cellulosic biofuels. Only three promoters have been commonly used for gene expression in C. thermocellum, gapDH, cbp and eno. Of the new promoters tested, 2638, 2926, 966 and 815 showed reliable expression. The 2638 promoter showed relatively higher activity when driving adhB (compared to lacZ), and the 815 promoter showed relatively higher activity when driving lacZ (compared to adhB).

11.
Circulation ; 130(11): 902-9, 2014 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-24982127

RESUMO

BACKGROUND: Regulation of vascular endothelial growth factor receptor-2 (VEGFR2) signaling is a control point that determines the extent of vascular tree formation. Recent studies demonstrated an important role played by VEGFR2 endothelial trafficking in control of its activity and suggested the involvement of a phosphotyrosine phosphatase 1b (PTP1b) in this process. This study was designed to define the role of PTP1b in endothelial VEGFR2 signaling and its role in regulation of angiogenesis and arteriogenesis. METHODS AND RESULTS: We generated mice carrying an endothelial-specific deletion of PTP1b and examined the effect of this knockout on VEGF signaling, angiogenesis, and arteriogenesis in vitro and in vivo. PTP1b knockout endothelial cells had increased VEGF-dependent activation of extracellular signal-regulated kinase signaling, sprouting, migration, and proliferation compared with controls. Endothelial PTP1b null mice had increased retinal and Matrigel implant angiogenesis and accelerated wound healing, pointing to enhanced angiogenesis. Increased arteriogenesis was demonstrated by observations of faster recovery of arterial blood flow and large numbers of newly formed arterioles in the hindlimb ischemia mouse model. PTP1b endothelial knockout also rescued impaired blood flow recovery after common femoral artery ligation in synectin null mice. CONCLUSIONS: PTP1b is a key regulator of endothelial VEGFR2 signaling and plays an important role in regulation of the extent of vascular tree formation.


Assuntos
Células Endoteliais/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Transdução de Sinais/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Aorta/citologia , Movimento Celular/fisiologia , Proliferação de Células , Modelos Animais de Doenças , Células Endoteliais/citologia , Feminino , Membro Posterior/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana , Isquemia/metabolismo , Isquemia/fisiopatologia , Masculino , Camundongos , Camundongos Mutantes , Neovascularização Fisiológica/fisiologia , Cultura Primária de Células , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , RNA Interferente Pequeno/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
12.
J Biol Chem ; 289(1): 510-9, 2014 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-24235146

RESUMO

Angiopoietin-2 (Ang2) is an extracellular protein and one of the principal ligands of Tie2 receptor that is involved in the regulation of vascular integrity, quiescence, and inflammation. The mode of secretion of Ang2 has never been established, however. Here, we provide evidence that Ang2 is secreted from endothelial cells via exosomes and that this process is inhibited by the PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling pathway, whereas it is positively regulated by the syndecan-4/syntenin pathway. Vascular defects in Akt1 null mice arise, in part, because of excessive Ang2 secretion and can be rescued by the syndecan-4 knock-out that reduces extracellular Ang2 levels. This novel mechanism connects three critical signaling pathways: angiopoietin/Tie2, PI3K/Akt/eNOS, and syndecan/syntenin, which play important roles in vascular growth and stabilization.


Assuntos
Angiopoietina-2/metabolismo , Células Endoteliais/metabolismo , Exossomos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , Sindecana-4/metabolismo , Sinteninas/metabolismo , Angiopoietina-2/genética , Animais , Células Cultivadas , Células Endoteliais/citologia , Exossomos/genética , Camundongos , Camundongos Knockout , Neovascularização Fisiológica/fisiologia , Óxido Nítrico Sintase Tipo III/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Sindecana-4/genética , Sinteninas/genética
13.
Circ Res ; 113(9): 1076-86, 2013 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-23897694

RESUMO

RATIONALE: Arteriogenesis is the process of formation of arterial conduits. Its promotion is an attractive therapeutic strategy in occlusive atherosclerotic diseases. Despite the functional and clinical importance of arteriogenesis, the biology of the process is poorly understood. Synectin, a gene previously implicated in the regulation of vascular endothelial cell growth factor signaling, offers a unique opportunity to determine relative contributions of various cell types to arteriogenesis. OBJECTIVE: We investigated the cell-autonomous effects of a synectin knockout in arterial morphogenesis. METHODS AND RESULTS: A floxed synectin knockin mouse line was crossbred with endothelial-specific (Tie2, Cdh5, Pdgfb) and smooth muscle myosin heavy chain-specific Cre driver mouse lines to produce cell type-specific deletions. Ablation of synectin expression in endothelial, but not smooth muscle cells resulted in the presence of developmental arterial morphogenetic defects (smaller size of the arterial tree, reduced number of arterial branches and collaterals) and impaired arteriogenesis in adult mice. CONCLUSIONS: Synectin modulates developmental and adult arteriogenesis in an endothelial cell-autonomous fashion. These findings show for the first time that endothelial cells are central to both developmental and adult arteriogenesis and provide a model for future studies of factors involved in this process.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Artérias/metabolismo , Células Endoteliais/metabolismo , Neovascularização Fisiológica , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células Cultivadas , Genótipo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fenótipo , Fatores de Tempo
15.
Cancer Cell ; 23(4): 477-88, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23597562

RESUMO

Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the pharmacologic characterization of the chemical SSR128129E (SSR), which inhibits fibroblast growth factor receptor (FGFR) signaling by binding to the extracellular FGFR domain without affecting orthosteric FGF binding. SSR exhibits allosteric properties, including probe dependence, signaling bias, and ceiling effects. Inhibition by SSR is highly conserved throughout the animal kingdom. Oral delivery of SSR inhibits arthritis and tumors that are relatively refractory to anti-vascular endothelial growth factor receptor-2 antibodies. Thus, orally-active extracellularly acting small-molecule modulators of RTKs with allosteric properties can be developed and may offer opportunities to improve anticancer treatment.


Assuntos
Inibidores de Proteínas Quinases/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Regulação Alostérica , Animais , Anticorpos Monoclonais/farmacologia , Artrite Experimental/tratamento farmacológico , Reabsorção Óssea/tratamento farmacológico , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/metabolismo , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Neovascularização Patológica/tratamento farmacológico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Transdução de Sinais , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Circulation ; 126(22): 2589-600, 2012 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-23091063

RESUMO

BACKGROUND: Arteriogenesis and collateral formation are complex processes requiring integration of multiple inputs to coordinate vessel branching, growth, maturation, and network size. Factors regulating these processes have not been determined. METHODS AND RESULTS: We used an inhibitor of NFκB activation (IκBαSR) under control of an endothelial-specific inducible promoter to selectively suppress endothelial nuclear factor-κB activation during development, in the adult vasculature, or in vitro. Inhibition of nuclear factor-κB activation resulted in formation of an excessively branched arterial network that was composed of immature vessels and provided poor distal tissue perfusion. Molecular analysis demonstrated reduced adhesion molecule expression leading to decreased monocyte influx, reduced hypoxia-inducible factor-1α levels, and a marked decrease in δ-like ligand 4 expression with a consequent decrease in Notch signaling. The latter was the principal cause of increased vascular branching as treatment with Jagged-1 peptide reduced the size of the arterial network to baseline levels. CONCLUSIONS: These findings identify nuclear factor-κB as a key regulator of adult and developmental arteriogenesis and collateral formation. Nuclear factor-κB achieves this by regulating hypoxia-inducible factor-1α-dependent expression of vascular endothelial growth factor-A and platelet-derived growth factor-BB, which are necessary for the development and maturation of the arterial collateral network, and by regulating δ-like ligand 4 expression, which in turn determines the size and complexity of the network.


Assuntos
Células Endoteliais/metabolismo , Isquemia/fisiopatologia , Subunidade p50 de NF-kappa B/metabolismo , Neovascularização Patológica/fisiopatologia , Neovascularização Fisiológica/fisiologia , Animais , Animais Recém-Nascidos , Becaplermina , Encéfalo/metabolismo , Modelos Animais de Doenças , Membro Posterior/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isquemia/metabolismo , Camundongos , Camundongos Transgênicos , Subunidade p50 de NF-kappa B/genética , Neovascularização Patológica/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
17.
PLoS One ; 7(5): e37600, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22629427

RESUMO

BACKGROUND: The fibroblast growth factor (FGF) system plays a critical role in the maintenance of vascular integrity via enhancing the stability of VE-cadherin at adherens junctions. However, the precise molecular mechanism is not well understood. In the present study, we aimed to investigate the detailed mechanism of FGF regulation of VE-cadherin function that leads to endothelial junction stabilization. METHODS AND FINDINGS: In vitro studies demonstrated that the loss of FGF signaling disrupts the VE-cadherin-catenin complex at adherens junctions by increasing tyrosine phosphorylation levels of VE-cadherin. Among protein tyrosine phosphatases (PTPs) known to be involved in the maintenance of the VE-cadherin complex, suppression of FGF signaling reduces SHP2 expression levels and SHP2/VE-cadherin interaction due to accelerated SHP2 protein degradation. Increased endothelial permeability caused by FGF signaling inhibition was rescued by SHP2 overexpression, indicating the critical role of SHP2 in the maintenance of endothelial junction integrity. CONCLUSIONS: These results identify FGF-dependent maintenance of SHP2 as an important new mechanism controlling the extent of VE-cadherin tyrosine phosphorylation, thereby regulating its presence in adherens junctions and endothelial permeability.


Assuntos
Junções Aderentes/metabolismo , Antígenos CD/metabolismo , Caderinas/metabolismo , Células Endoteliais/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Transdução de Sinais/fisiologia , Animais , Permeabilidade Capilar , Bovinos , Células Cultivadas , Células Endoteliais/citologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Fosforilação
18.
Dev Cell ; 18(5): 713-24, 2010 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-20434959

RESUMO

VEGF is the key growth factor regulating arterial morphogenesis. However, molecular events involved in this process have not been elucidated. Synectin null mice demonstrate impaired VEGF signaling and a marked reduction in arterial morphogenesis. Here, we show that this occurs due to delayed trafficking of VEGFR2-containing endosomes that exposes internalized VEGFR2 to selective dephosphorylation by PTP1b on Y(1175) site. Synectin involvement in VEGFR2 intracellular trafficking requires myosin-VI, and myosin-VI knockout in mice or knockdown in zebrafish phenocopy the synectin null phenotype. Silencing of PTP1b restores VEGFR2 activation and significantly recovers arterial morphogenesis in myosin-VI(-/-) knockdown zebrafish and synectin(-/-) mice. We conclude that activation of the VEGF-mediated arterial morphogenesis cascade requires phosphorylation of the VEGFR2 Y(1175) site that is dependent on trafficking of internalized VEGFR2 away from the plasma membrane via a synectin-myosin-VI complex. This key event in VEGF signaling occurs at an intracellular site and is regulated by a novel endosomal trafficking-dependent process.


Assuntos
Artérias/embriologia , Endotélio Vascular/fisiologia , Morfogênese/fisiologia , Neuropeptídeos/deficiência , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Artérias/crescimento & desenvolvimento , Proteínas de Transporte/genética , Membrana Celular/fisiologia , Endocitose , Endotélio Vascular/embriologia , Inativação Gênica , Camundongos , Camundongos Knockout , Cadeias Pesadas de Miosina/deficiência , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/fisiologia , Neuropeptídeos/genética , Fosforilação , Fator A de Crescimento do Endotélio Vascular/fisiologia , Peixe-Zebra
19.
J Clin Invest ; 120(4): 1217-28, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20237411

RESUMO

Arterial morphogenesis is an important and poorly understood process. In particular, the signaling events controlling arterial formation have not been established. We evaluated whether alterations in the balance between ERK1/2 and PI3K signaling pathways could stimulate arterial formation in the setting of defective arterial morphogenesis in mice and zebrafish. Increased ERK1/2 activity in mouse ECs with reduced VEGF responsiveness was achieved in vitro and in vivo by downregulating PI3K activity, suppressing Akt1 but not Akt2 expression, or introducing a constitutively active ERK1/2 construct. Such restoration of ERK1/2 activation was sufficient to restore impaired arterial development and branching morphogenesis in synectin-deficient mice and synectin-knockdown zebrafish. The same approach effectively stimulated arterial growth in adult mice, restoring arteriogenesis in mice lacking synectin and in atherosclerotic mice lacking both LDL-R and ApoB48. We therefore conclude that PI3K-ERK1/2 crosstalk plays a key role in the regulation of arterial growth and that the augmentation of ERK signaling via suppression of the PI3K signaling pathway can effectively stimulate arteriogenesis.


Assuntos
Artérias/crescimento & desenvolvimento , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Morfogênese , Proteínas Proto-Oncogênicas c-akt/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/fisiologia , Masculino , Camundongos , Neovascularização Fisiológica , Neuropeptídeos/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra
20.
PLoS One ; 5(12): e15581, 2010 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-21209904

RESUMO

GIPC1 is a cytoplasmic scaffold protein that interacts with numerous receptor signaling complexes, and emerging evidence suggests that it plays a role in tumorigenesis. GIPC1 is highly expressed in a number of human malignancies, including breast, ovarian, gastric, and pancreatic cancers. Suppression of GIPC1 in human pancreatic cancer cells inhibits in vivo tumor growth in immunodeficient mice. To better understand GIPC1 function, we suppressed its expression in human breast and colorectal cancer cell lines and human mammary epithelial cells (HMECs) and assayed both gene expression and cellular phenotype. Suppression of GIPC1 promotes apoptosis in MCF-7, MDA-MD231, SKBR-3, SW480, and SW620 cells and impairs anchorage-independent colony formation of HMECs. These observations indicate GIPC1 plays an essential role in oncogenic transformation, and its expression is necessary for the survival of human breast and colorectal cancer cells. Additionally, a GIPC1 knock-down gene signature was used to interrogate publically available breast and ovarian cancer microarray datasets. This GIPC1 signature statistically correlates with a number of breast and ovarian cancer phenotypes and clinical outcomes, including patient survival. Taken together, these data indicate that GIPC1 inhibition may represent a new target for therapeutic development for the treatment of human cancers.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Apoptose , Neoplasias da Mama/genética , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica , Neoplasias Colorretais/metabolismo , Progressão da Doença , Células Epiteliais/citologia , Feminino , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , Interferência de RNA
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