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1.
J Cell Sci ; 136(10)2023 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-37226882

RESUMO

Vascular endothelial growth factor receptor 2 (VEGFR2, encoded by KDR) regulates endothelial function and angiogenesis. VEGFR2 undergoes ubiquitination that programs this receptor for trafficking and proteolysis, but the ubiquitin-modifying enzymes involved are ill-defined. Herein, we used a reverse genetics screen for the human E2 family of ubiquitin-conjugating enzymes to identify gene products that regulate VEGFR2 ubiquitination and proteolysis. We found that depletion of either UBE2D1 or UBE2D2 in endothelial cells caused a rise in steady-state VEGFR2 levels. This rise in plasma membrane VEGFR2 levels impacted on VEGF-A-stimulated signalling, with increased activation of canonical MAPK, phospholipase Cγ1 and Akt pathways. Analysis of biosynthetic VEGFR2 is consistent with a role for UBE2D enzymes in influencing plasma membrane VEGFR2 levels. Cell-surface-specific biotinylation and recycling studies showed an increase in VEGFR2 recycling to the plasma membrane upon reduction in UBE2D levels. Depletion of either UBE2D1 or UBE2D2 stimulated endothelial tubulogenesis, which is consistent with increased VEGFR2 plasma membrane levels promoting the cellular response to exogenous VEGF-A. Our studies identify a key role for UBE2D1 and UBE2D2 in regulating VEGFR2 function in angiogenesis.


Assuntos
Células Endoteliais , Enzimas de Conjugação de Ubiquitina , Humanos , Enzimas de Conjugação de Ubiquitina/genética , Fator A de Crescimento do Endotélio Vascular , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Ubiquitinação
2.
Methods Mol Biol ; 2475: 113-124, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35451752

RESUMO

The endothelial response to vascular endothelial growth factor A (VEGF-A) regulates many aspects of animal physiology in health and disease. Such VEGF-A-regulated phenomena include vasculogenesis, angiogenesis, tumor growth and progression. VEGF-A binding to receptor tyrosine kinases such as vascular endothelial growth factor receptor 2 (VEGFR2 ) activates multiple signal transduction pathways and changes in homeostasis, metabolism, gene expression, cell proliferation, migration, and survival. One such VEGF-A-regulated response is a rapid rise in cytosolic calcium ion levels which modulates different biochemical events and impacts on endothelial-specific responses. Here, we present a series of detailed and robust protocols for evaluating ligand-stimulated cytosolic calcium ion flux in endothelial cells. By monitoring an endogenous endothelial transcription factor (NFATc2 ) which displays calcium-sensitive redistribution, we can assess the relevance of cytosolic calcium to protein function. This protocol can be easily applied to both adherent and non-adherent cultured cells to evaluate calcium ion flux in response to exogenous stimuli such as VEGF-A.


Assuntos
Células Endoteliais , Fator A de Crescimento do Endotélio Vascular , Animais , Cálcio/metabolismo , Movimento Celular , Células Cultivadas , Células Endoteliais/metabolismo , Neovascularização Fisiológica/fisiologia , Fosforilação , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
3.
Chembiochem ; 23(1): e202100463, 2022 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-34647407

RESUMO

Vascular endothelial growth factors (VEGFs) regulate significant pathways in angiogenesis, myocardial and neuronal protection, metabolism, and cancer progression. The VEGF-B growth factor is involved in cell survival, anti-apoptotic and antioxidant mechanisms, through binding to VEGF receptor 1 and neuropilin-1 (NRP1). We employed surface plasmon resonance technology and X-ray crystallography to analyse the molecular basis of the interaction between VEGF-B and the b1 domain of NRP1, and developed VEGF-B C-terminus derived peptides to be used as chemical tools for studying VEGF-B - NRP1 related pathways. Peptide lipidation was used as a means to stabilise the peptides. VEGF-B-derived peptides containing a C-terminal arginine show potent binding to NRP1-b1. Peptide lipidation increased binding residence time and improved plasma stability. A crystal structure of a peptide with NRP1 demonstrated that VEGF-B peptides bind at the canonical C-terminal arginine binding site. VEGF-B C-terminus imparts higher affinity for NRP1 than the corresponding VEGF-A165 region. This tight binding may impact on the activity and selectivity of the full-length protein. The VEGF-B167 derived peptides were more effective than VEGF-A165 peptides in blocking functional phosphorylation events. Blockers of VEGF-B function have potential applications in diabetes and non-alcoholic fatty liver disease.


Assuntos
Neuropilina-1/metabolismo , Peptídeos/metabolismo , Fator B de Crescimento do Endotélio Vascular/metabolismo , Humanos , Neuropilina-1/química , Peptídeos/química , Ligação Proteica , Fator B de Crescimento do Endotélio Vascular/química
4.
J Med Chem ; 61(9): 4135-4154, 2018 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-29648813

RESUMO

We report the design, synthesis, and biological evaluation of some potent small-molecule neuropilin-1 (NRP1) antagonists. NRP1 is implicated in the immune response to tumors, particularly in Treg cell fragility, required for PD1 checkpoint blockade. The design of these compounds was based on a previously identified compound EG00229. The design of these molecules was informed and supported by X-ray crystal structures. Compound 1 (EG01377) was identified as having properties suitable for further investigation. Compound 1 was then tested in several in vitro assays and was shown to have antiangiogenic, antimigratory, and antitumor effects. Remarkably, 1 was shown to be selective for NRP1 over the closely related protein NRP2. In purified Nrp1+, FoxP3+, and CD25+ populations of Tregs from mice, 1 was able to block a glioma-conditioned medium-induced increase in TGFß production. This comprehensive characterization of a small-molecule NRP1 antagonist provides the basis for future in vivo studies.


Assuntos
Imunomodulação/efeitos dos fármacos , Neuropilina-1/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/biossíntese , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Desenho de Fármacos , Humanos , Camundongos , Modelos Moleculares , Conformação Molecular , Ácidos Pentanoicos/química , Ácidos Pentanoicos/farmacologia , Bibliotecas de Moléculas Pequenas/química , Linfócitos T Reguladores/imunologia , Fator A de Crescimento do Endotélio Vascular/farmacologia
5.
Mol Cell Proteomics ; 16(2): 168-180, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28007913

RESUMO

p130Cas is a polyvalent adapter protein essential for cardiovascular development, and with a key role in cell movement. In order to identify the pathways by which p130Cas exerts its biological functions in endothelial cells we mapped the p130Cas interactome and its dynamic changes in response to VEGF using high-resolution mass spectrometry and reconstruction of protein interaction (PPI) networks with the aid of multiple PPI databases. VEGF enriched the p130Cas interactome in proteins involved in actin cytoskeletal dynamics and cell movement, including actin-binding proteins, small GTPases and regulators or binders of GTPases. Detailed studies showed that p130Cas association of the GTPase-binding scaffold protein, IQGAP1, plays a key role in VEGF chemotactic signaling, endothelial polarization, VEGF-induced cell migration, and endothelial tube formation. These findings indicate a cardinal role for assembly of the p130Cas interactome in mediating the cell migratory response to VEGF in angiogenesis, and provide a basis for further studies of p130Cas in cell movement.


Assuntos
Quimiotaxia/efeitos dos fármacos , Proteína Substrato Associada a Crk/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Proteômica/métodos , Fator A de Crescimento do Endotélio Vascular/farmacologia , Bases de Dados de Proteínas , Células Endoteliais da Veia Umbilical Humana , Humanos , Espectrometria de Massas , Mapas de Interação de Proteínas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
6.
Sci Rep ; 6: 27378, 2016 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-27293031

RESUMO

Imatinib was the first targeted tyrosine kinase inhibitor to be approved for clinical use, and remains first-line therapy for Philadelphia chromosome (Ph+)-positive chronic myelogenous leukaemia. We show that treatment of human glioblastoma multiforme (GBM) tumour cells with imatinib and the closely-related drug, nilotinib, strikingly increases tyrosine phosphorylation of p130Cas, focal adhesion kinase (FAK) and the downstream adaptor protein paxillin (PXN), resulting in enhanced cell migration and invasion. Imatinib and nilotinib-induced tyrosine phosphorylation was dependent on expression of p130Cas and FAK activity and was independent of known imatinib targets including Abl, platelet derived growth factor receptor beta (PDGFRß) and the collagen receptor DDR1. Imatinib and nilotinib treatment increased two dimensional cell migration and three dimensional radial spheroid invasion in collagen. In addition, silencing of p130Cas and inhibition of FAK activity both strongly reduced imatinib and nilotinib stimulated invasion. Importantly, imatinib and nilotinib increased tyrosine phosphorylation of p130Cas, FAK, PXN and radial spheroid invasion in stem cell lines isolated from human glioma biopsies. These findings identify a novel mechanism of action in GBM cells for two well established front line therapies for cancer resulting in enhanced tumour cell motility.


Assuntos
Proteína Substrato Associada a Crk/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Glioblastoma/tratamento farmacológico , Mesilato de Imatinib/farmacologia , Invasividade Neoplásica/patologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Pirimidinas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Receptor com Domínio Discoidina 1/metabolismo , Glioblastoma/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Paxilina/metabolismo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos
7.
Biol Reprod ; 94(6): 142, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27103444

RESUMO

Uterine artery (UtA) adenovirus (Ad) vector-mediated overexpression of vascular endothelial growth factor (VEGF) enhances uterine blood flow in normal sheep pregnancy and increases fetal growth in the overnourished adolescent sheep model of fetal growth restriction (FGR). Herein, we examined its impact on gestation length, neonatal survival, early postnatal growth and metabolism. Singleton-bearing ewes were evenly allocated to receive Ad.VEGF-A165 (5 × 10(10) particles/ml, 10 ml, n = 17) or saline (10 ml, n = 16) injected into each UtA at laparotomy (0.6 gestation). Fetal growth was serially monitored (blind) by ultrasound until delivery. Lambs were weighed and blood was sampled weekly and a glucose tolerance test performed (68-day postnatal age). Hepatic DNA/RNA was extracted at necropsy (83-day postnatal age) to examine methylation status of eight somatotropic axis genes. IGF1 mRNA and protein expression were measured by RT-PCR and radioimmunoassay, respectively. All pregnancies remained viable following Ad.VEGF-A165 treatment. Fetal abdominal circumference and renal volume were greater in the Ad.VEGF-A165 group compared with the saline group at 21/28 days (P ≤ 0.04) postinjection. At delivery, gestation length (P = 0.07), lamb birthweight (P = 0.08), umbilical girth (P = 0.06), and plasma glucose (P = 0.09) tended to be greater in Ad.VEGF-A165-treated lambs. Levels of neonatal intervention required to ensure survival was equivalent between groups. Absolute postnatal growth rate (P = 0.02), insulin area under the curve (P = 0.04) and carcass weight at necropsy (P = 0.04) were increased by Ad.VEGF-A165 treatment. There was no impact on markers of insulin sensitivity or methylation/expression of key genes involved in somatic growth. Ad.VEGF-A165 gene therapy increased fetal growth in a sheep FGR model, and lambs continued to thrive during the neonatal and early postnatal period.


Assuntos
Retardo do Crescimento Fetal/terapia , Terapia Genética , Fator A de Crescimento do Endotélio Vascular/genética , Adenoviridae , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Composição Corporal , Metilação de DNA , Feminino , Desenvolvimento Fetal , Teste de Tolerância a Glucose , Gravidez , Resultado da Gravidez , Ovinos
8.
Oncotarget ; 7(9): 9801-14, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26755653

RESUMO

Gliomas are the most commonly diagnosed primary tumors of the central nervous system (CNS). Median times of survival are dismal regardless of the treatment approach, underlying the need to develop more effective therapies. Modulation of the immune system is a promising strategy as innate and adaptive immunity play important roles in cancer progression. Glioma associated microglia and macrophages (GAMs) can comprise over 30% of the cells in glioma biopsies. Gliomas secrete cytokines that suppress the anti-tumorigenic properties of GAMs, causing them to secrete factors that support the tumor's spread and growth. Neuropilin 1 (Nrp1) is a transmembrane receptor that in mice both amplifies pro-angiogenic signaling in the tumor microenvironment and affects behavior of innate immune cells. Using a Cre-lox system, we generated mice that lack expression of Nrp1 in GAMs. We demonstrate, using an in vivo orthotopic glioma model, that tumors in mice with Nrp1-deficient GAMs exhibit less vascularity, grow at a slower pace, and are populated by increased numbers of anti-tumorigenic GAMs. Moreover, glioma survival times in mice with Nrp1-deficient GAMs were significantly longer. Treating wild-type mice with a small molecule inhibitor of Nrp1's b1 domain, EG00229, which we show here is selective for Nrp1 over Nrp2, yielded an identical outcome. Nrp1-deficient or EG00229-treated wild-type microglia exhibited a shift towards anti-tumorigenicity as evident by altered inflammatory marker profiles in vivo and decreased SMAD2/3 activation when conditioned in the presence of glioma-derived factors. These results provide support for the proposal that pharmacological inhibition of Nrp1 constitutes a potential strategy for suppressing glioma progression.


Assuntos
Antineoplásicos/uso terapêutico , Glioma/tratamento farmacológico , Macrófagos/imunologia , Microglia/patologia , Neuropilina-1 , Animais , Linhagem Celular Tumoral , Progressão da Doença , Glioma/mortalidade , Glioma/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neuropilina-1/antagonistas & inibidores , Neuropilina-1/deficiência , Neuropilina-1/genética , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo
9.
Mol Biol Cell ; 25(16): 2509-21, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24966171

RESUMO

Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology. VEGF-A stimulates signal transduction pathways that modulate endothelial outputs such as cell migration, proliferation, tubulogenesis, and cell-cell interactions. Multiple VEGF-A isoforms exist, but the biological significance of this is unclear. Here we analyzed VEGF-A isoform-specific stimulation of VCAM-1 gene expression, which controls endothelial-leukocyte interactions, and show that this is dependent on both ERK1/2 and activating transcription factor-2 (ATF-2). VEGF-A isoforms showed differential ERK1/2 and p38 MAPK phosphorylation kinetics. A key feature of VEGF-A isoform-specific ERK1/2 activation and nuclear translocation was increased phosphorylation of ATF-2 on threonine residue 71 (T71). Using reverse genetics, we showed ATF-2 to be functionally required for VEGF-A-stimulated endothelial VCAM-1 gene expression. ATF-2 knockdown blocked VEGF-A-stimulated VCAM-1 expression and endothelial-leukocyte interactions. ATF-2 was also required for other endothelial cell outputs, such as cell migration and tubulogenesis. In contrast, VCAM-1 was essential only for promoting endothelial-leukocyte interactions. This work presents a new paradigm for understanding how soluble growth factor isoforms program complex cellular outputs and responses by modulating signal transduction pathways.


Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Leucócitos/metabolismo , Sistema de Sinalização das MAP Quinases , Molécula 1 de Adesão de Célula Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator 2 Ativador da Transcrição/genética , Movimento Celular , Proliferação de Células , Expressão Gênica , Humanos , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
10.
J Cell Sci ; 127(Pt 12): 2647-58, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24762811

RESUMO

DOK1 regulates platelet-derived growth factor (PDGF)-BB-stimulated glioma cell motility. Mechanisms regulating tumour cell motility are essential for invasion and metastasis. We report here that PDGF-BB-mediated glioma cell invasion and migration are dependent on the adaptor protein downstream of kinase 1 (DOK1). DOK1 is expressed in several glioma cell lines and in tumour biopsies from high-grade gliomas. DOK1 becomes tyrosine phosphorylated upon PDGF-BB stimulation of human glioma cells. Knockdown of DOK1 or expression of a DOK1 mutant (DOK1FF) containing Phe in place of Tyr at residues 362 and 398, resulted in inhibition of both the PDGF-BB-induced tyrosine phosphorylation of p130Cas (also known as BCAR1) and the activation of Rap1. DOK1 colocalises with tyrosine phosphorylated p130Cas at the cell membrane of PDGF-BB-treated cells. Expression of a non-tyrosine-phosphorylatable substrate domain mutant of p130Cas (p130Cas15F) inhibited PDGF-BB-mediated Rap1 activation. Knockdown of DOK1 and Rap1 inhibited PDGF-BB-induced chemotactic cell migration, and knockdown of DOK1 and Rap1 and expression of DOK1FF inhibited PDGF-mediated three-dimensional (3D) spheroid invasion. These data show a crucial role for DOK1 in the regulation of PDGF-BB-mediated tumour cell motility through a p130Cas-Rap1 signalling pathway. [Corrected]


Assuntos
Neoplasias Encefálicas/metabolismo , Proteína Substrato Associada a Crk/metabolismo , Proteínas de Ligação a DNA/fisiologia , Glioblastoma/metabolismo , Fosfoproteínas/fisiologia , Proteínas Proto-Oncogênicas c-sis/fisiologia , Proteínas de Ligação a RNA/fisiologia , Proteínas de Ligação a Telômeros/metabolismo , Becaplermina , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Quimiotaxia , Glioblastoma/patologia , Humanos , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Complexo Shelterina , Transdução de Sinais , Quinases da Família src/metabolismo
11.
Chembiochem ; 15(8): 1161-70, 2014 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-24771685

RESUMO

The interaction between VEGF-A and its neuropilin (NRP) receptors mediates a number of important biological effects. NRP1 and the related molecule NRP2 are widely expressed on multiple tumour types and throughout the tumour vasculature, and are emerging as critical molecules required for the progression of angiogenic diseases. Given the increasing evidence supporting a role for NRP1 in tumour development, there is growing interest in developing inhibitors of NRP1 interactions with VEGF and its other ligands. In order to probe the interaction we synthesised a number of exon 7- and 8-derived bicyclic peptides with N-terminal lipophilic groups and found a simple N-octanoyl derivative (EG00086) to be the most potent and functionally active. Detailed modelling studies indicated that new intramolecular hydrogen bonds were formed, stabilising the structure and possibly contributing to the potency. Removal of a salt bridge between D142 and R164 implicated in VEGF-A binding to neuropilin-1 had a minor effect on potency. Isothermal calorimetry was used to assess binding of EG00086 to NRP1 and NRP2, and the stability of the peptide in serum and in vivo was investigated. EG00086 is a potent blocker of VEGF-promoted cellular adhesion to extracellular matrices, and phosphorylation of p130Cas contributes to this effect.


Assuntos
Neuropilina-1/metabolismo , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/metabolismo , Sítios de Ligação , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteína Substrato Associada a Crk/metabolismo , Éxons/genética , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Lipopeptídeos/química , Lipopeptídeos/metabolismo , Lipopeptídeos/farmacologia , Simulação de Dinâmica Molecular , Neuropilina-1/química , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/genética
12.
Hum Gene Ther ; 25(4): 375-84, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24593228

RESUMO

Fetal growth restriction (FGR) occurs in ∼8% of pregnancies and is a major cause of perinatal mortality and morbidity. There is no effective treatment. FGR is characterized by reduced uterine blood flow (UBF). In normal sheep pregnancies, local uterine artery (UtA) adenovirus (Ad)-mediated overexpression of vascular endothelial growth factor (VEGF) increases UBF. Herein we evaluated Ad.VEGF therapy in the overnourished adolescent ewe, an experimental paradigm in which reduced UBF from midgestation correlates with reduced lamb birthweight near term. Singleton pregnancies were established using embryo transfer in adolescent ewes subsequently offered a high intake (n=45) or control intake (n=12) of a complete diet to generate FGR or normal fetoplacental growth, respectively. High-intake ewes were randomized midgestation to receive bilateral UtA injections of 5×10¹¹ particles Ad.VEGF-A165 (n=18), control vector Ad.LacZ (n=14), or control saline (n=13). Fetal growth/well-being were evaluated using serial ultrasound. UBF was monitored using indwelling flowprobes until necropsy at 0.9 gestation. Vasorelaxation, neovascularization within the perivascular adventitia, and placental mRNA expression of angiogenic factors/receptors were examined using organ bath analysis, anti-vWF immunohistochemistry, and qRT-PCR, respectively. Ad.VEGF significantly increased ultrasonographic fetal growth velocity at 3-4 weeks postinjection (p=0.016-0.047). At 0.9 gestation fewer fetuses were markedly growth-restricted (birthweight >2SD below contemporaneous control-intake mean) after Ad.VEGF therapy. There was also evidence of mitigated fetal brain sparing (lower biparietal diameter-to-abdominal circumference and brain-to-liver weight ratios). No effects were observed on UBF or neovascularization; however, Ad.VEGF-transduced vessels demonstrated strikingly enhanced vasorelaxation. Placental efficiency (fetal-to-placental weight ratio) and FLT1/KDR mRNA expression were increased in the maternal but not fetal placental compartments, suggesting downstream effects on placental function. Ad.VEGF gene therapy improves fetal growth in a sheep model of FGR, although the precise mechanism of action remains unclear.


Assuntos
Adenoviridae/genética , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/terapia , Vetores Genéticos/genética , Placenta/metabolismo , Útero/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Feminino , Retardo do Crescimento Fetal/diagnóstico por imagem , Expressão Gênica , Terapia Genética , Vetores Genéticos/administração & dosagem , Neovascularização Fisiológica , Circulação Placentária , Gravidez , Fluxo Sanguíneo Regional , Transdução Genética , Ultrassonografia , Artéria Uterina
13.
Cell Signal ; 25(4): 766-77, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23277200

RESUMO

p130Cas/breast cancer anti-oestrogen resistance 1 (BCAR1) is a member of the Cas (Crk-associated substrate) family of adaptor proteins, which have emerged as key signalling nodes capable of interactions with multiple proteins, with important regulatory roles in normal and pathological cell function. The Cas family of proteins is characterised by the presence of multiple conserved motifs for protein-protein interactions, and by extensive tyrosine and serine phosphorylations. Recent studies show that p130Cas contributes to migration, cell cycle control and apoptosis. p130Cas is essential during early embryogenesis, with a critical role in cardiovascular development. Furthermore, p130Cas has been reported to be involved in the development and progression of several human cancers. p130Cas is able to perform roles in multiple processes due to its capacity to regulate a diverse array of signalling pathways, transducing signals from growth factor receptor tyrosine kinases, non-receptor tyrosine kinases, and integrins. In this review we summarise the current understanding of the structure, function, and regulation of p130Cas, and discuss the importance of p130Cas in both physiological and pathophysiological settings, with a focus on the cardiovascular system and cancer.


Assuntos
Proteína Substrato Associada a Crk/metabolismo , Transdução de Sinais , Sistema Cardiovascular/metabolismo , Proteína Substrato Associada a Crk/análise , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Mecanotransdução Celular , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação
14.
Biochem Soc Trans ; 39(6): 1583-91, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22103491

RESUMO

Essential roles of NRP1 (neuropilin-1) in cardiovascular development and in neuronal axon targeting during embryogenesis are thought to be mediated primarily through binding of NRP1 to two unrelated types of ligands: the VEGF (vascular endothelial growth factor) family of angiogenic cytokines in the endothelium, and the class 3 semaphorins in neurons. A widely accepted mechanism for the role of NRP1 in the endothelium is VEGF binding to NRP1 and VEGFR2 (VEGF receptor 2) and VEGF-dependent formation of complexes or NRP1-VEGFR2 holoreceptors with enhanced signalling activity and biological function. However, although some basic features of this model are solidly based on biochemical and cellular data, others are open to question. Furthermore, a mechanistic account of NRP1 has to accommodate research which emphasizes the diversity of NRP1 functions in different cell types and particularly an emerging role in signalling by other growth factor ligands for RTKs (receptor tyrosine kinases) such as HGF (hepatocyte growth factor) and PDGF (platelet-derived growth factor). It is uncertain, however, whether the model of NRP1-RTK heterocomplex formation applies in all of these situations. In the light of these developments, the need to explain mechanistically the role of NRP1 in signalling is coming increasingly to the fore. The present article focuses on some of the most important unresolved questions concerning the mechanism(s) through which NRP1 acts, and highlights recent findings which are beginning to generate insights into these questions.


Assuntos
Neuropilina-1/química , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais , Animais , Endotélio Vascular/metabolismo , Humanos , Neuropilina-1/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
15.
Mol Biol Cell ; 22(15): 2766-76, 2011 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-21653826

RESUMO

In endothelial cells, neuropilin-1 (NRP1) binds vascular endothelial growth factor (VEGF)-A and is thought to act as a coreceptor for kinase insert domain-containing receptor (KDR) by associating with KDR and enhancing VEGF signaling. Here we report mutations in the NRP1 b1 domain (Y297A and D320A), which result in complete loss of VEGF binding. Overexpression of Y297A and D320A NRP1 in human umbilical vein endothelial cells reduced high-affinity VEGF binding and migration toward a VEGF gradient, and markedly inhibited VEGF-induced angiogenesis in a coculture cell model. The Y297A NRP1 mutant also disrupted complexation between NRP1 and KDR and decreased VEGF-dependent phosphorylation of focal adhesion kinase at Tyr407, but had little effect on other signaling pathways. Y297A NRP1, however, heterodimerized with wild-type NRP1 and NRP2 indicating that nonbinding NRP1 mutants can act in a dominant-negative manner through formation of NRP1 dimers with reduced binding affinity for VEGF. These findings indicate that VEGF binding to NRP1 has specific effects on endothelial cell signaling and is important for endothelial cell migration and angiogenesis mediated via complex formation between NRP1 and KDR and increased signaling to focal adhesions. Identification of key residues essential for VEGF binding and biological functions provides the basis for a rational design of antagonists of VEGF binding to NRP1.


Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Neuropilina-1/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Sítios de Ligação , Adesão Celular , Movimento Celular , Técnicas de Cocultura , Dimerização , Células Endoteliais/citologia , Endotélio Vascular/citologia , Quinase 1 de Adesão Focal/genética , Humanos , Mutação , Neovascularização Fisiológica , Neuropilina-1/genética , Fosforilação , Plasmídeos , Ligação Proteica , Transdução de Sinais/fisiologia , Transfecção , Veias Umbilicais/citologia , Veias Umbilicais/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
16.
IUBMB Life ; 63(4): 258-63, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21488147

RESUMO

The Protein Kinase D (PKD) family comprises diacylglycerol stimulated serine/threonine protein kinases that participate in many key signaling pathways in a diverse range of cells. Recent studies show that PKD isoforms 1 and 2 play critical roles in vascular biology and angiogenesis and there has been considerable progress in determining some of the key angiogenic signaling pathways mediated by PKD in endothelial cells. Less is currently known regarding the specific roles of PKD isoforms in endothelial cells and the role of PKD in smooth muscle cells. PKD is also emerging as a potentially important mediator of tumor growth and tumor angiogenesis and there is growing interest in PKD as a novel therapeutic target in cancer.


Assuntos
Músculo Liso Vascular/fisiologia , Neovascularização Patológica/metabolismo , Proteína Quinase C/metabolismo , Animais , Células Endoteliais/metabolismo , Humanos , Isoenzimas/metabolismo , Músculo Liso Vascular/metabolismo , Neoplasias/irrigação sanguínea , Transdução de Sinais
17.
Biochem J ; 435(3): 609-18, 2011 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-21306301

RESUMO

NRP1 (neuropilin-1) is a co-receptor for members of the VEGF (vascular endothelial growth factor) family in endothelial cells, but is increasingly implicated in signalling induced by other growth factors. NRP1 is expressed in VSMCs (vascular smooth muscle cells), but its function and the mechanisms involved are poorly understood. The present study aimed to determine the role of NRP1 in the migratory response of HCASMCs (human coronary artery smooth muscle cells) to PDGF (platelet-derived growth factor), and to identify the signalling mechanisms involved. NRP1 is highly expressed in HAoSMCs (human aortic smooth muscle cells) and HCASMCs, and modified in VSMCs by CS (chondroitin sulfate)-rich O-linked glycosylation at Ser612. HCASMC migration induced by PDGF-BB and PDGF-AA was inhibited by NRP1 siRNA (small interfering RNA), and by adenoviral overexpression of an NRP1 mutant lacking the intracellular domain (Ad.NRP1ΔC). NRP1 co-immunoprecipitated with PDGFRα (PDGF receptor α), and immunofluorescent staining indicated that NRP1 and PDGFRα co-localized in VSMCs. NRP1 siRNA also inhibited PDGF-induced PDGFRα activation. NRP1-specific siRNA, Ad.NRP1ΔC and removal of CS glycans using chondroitinase all inhibited PDGF-BB and -AA stimulation of tyrosine phosphorylation of the adapter protein, p130Cas (Cas is Crk-associated substrate), with little effect on other major signalling pathways, and p130Cas knockdown inhibited HCASMC migration. Chemotaxis and p130Cas phosphorylation induced by PDGF were inhibited by chondroitinase, and, additionally, adenoviral expression of a non-glycosylatable NRP1S612A mutant inhibited chemotaxis, but not p130Cas phosphorylation. These results indicate a role for NRP1 and NRP1 glycosylation in mediating PDGF-induced VSMC migration, possibly by acting as a co-receptor for PDGFRα and via selective mobilization of a novel p130Cas tyrosine phosphorylation pathway.


Assuntos
Proteína Substrato Associada a Crk/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Neuropilina-1/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Adenoviridae , Movimento Celular , Células Cultivadas , Quimiotaxia , Proteína Substrato Associada a Crk/genética , Células Endoteliais , Humanos , Mutação , Miócitos de Músculo Liso/citologia , Neuropilina-1/genética , Fator de Crescimento Derivado de Plaquetas/genética
18.
Mol Cell Biol ; 31(6): 1174-85, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21245381

RESUMO

Neuropilin-1 (NRP1) is a receptor for vascular endothelial growth factor (VEGF) and plays an important role in mediating cell motility. However, the NRP1 signaling pathways important for cell motility are poorly understood. Here we report that p130(Cas) tyrosine phosphorylation is stimulated by hepatocyte growth factor and platelet-derived growth factor in U87MG glioma cells and VEGF in endothelial cells and is dependent on NRP1 via its intracellular domain. In endothelial cells, NRP1 silencing reduced, but did not prevent, VEGF receptor 2 (VEGFR2) phosphorylation, while expression of a mutant form of NRP1 lacking the intracellular domain (NRP1ΔC) did not affect receptor phosphorylation in U87MG cells or human umbilical vein endothelial cells (HUVECs). In HUVECs, NRP1 was also required for VEGF-induced phosphorylation of proline-rich tyrosine kinase 2, which was necessary for p130(Cas) phosphorylation. Importantly, knockdown of NRP1 or p130(Cas) or expression of either NRP1ΔC or a non-tyrosine-phosphorylatable substrate domain mutant protein (p130(Cas15F)) was sufficient to inhibit growth factor-mediated migration of glioma and endothelial cells. These data demonstrate for the first time the importance of the NRP1 intracellular domain in mediating a specific signaling pathway downstream of several receptor tyrosine kinases and identify a critical role for a novel NRP1-p130(Cas) pathway in the regulation of chemotaxis.


Assuntos
Movimento Celular , Proteína Substrato Associada a Crk/metabolismo , Células Endoteliais/metabolismo , Glioma/metabolismo , Neuropilina-1/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proteína Substrato Associada a Crk/genética , Células Endoteliais/citologia , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Glioma/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Mutação , Neuropilina-1/genética , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Tirosina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
19.
Biochem J ; 429(3): 565-72, 2010 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-20497126

RESUMO

VEGF (vascular endothelial growth factor) plays an essential role in angiogenesis during development and in disease largely mediated by signalling events initiated by binding of VEGF to its receptor, VEGFR2 (VEGF receptor 2)/KDR (kinase insert domain receptor). Recent studies indicate that VEGF activates PKD (protein kinase D) in endothelial cells to regulate a variety of cellular functions, including signalling events, proliferation, migration and angiogenesis. To better understand the role of PKD in VEGF-mediated endothelial function, we characterized the effects of a novel pyrazine benzamide PKD inhibitor CRT5 in HUVECs (human umbilical vein endothelial cells). The activity of the isoforms PKD1 and PKD2 were blocked by this inhibitor as indicated by reduced phosphorylation, at Ser916 and Ser876 respectively, after VEGF stimulation. The VEGF-induced phosphorylation of three PKD substrates, histone deacetylase 5, CREB (cAMP-response-element-binding protein) and HSP27 (heat-shock protein 27) at Ser82, was also inhibited by CRT5. In contrast, CRT6, an inactive analogue of CRT5, had no effect on PKD or HSP27 Ser82 phosphorylation. Furthermore, phosphorylation of HSP27 at Ser78, which occurs solely via the p38 MAPK (mitogen-activated protein kinase) pathway, was also unaffected by CRT5. In vitro kinase assays show that CRT5 did not significantly inhibit several PKC isoforms expressed in endothelial cells. CRT5 also decreased VEGF-induced endothelial migration, proliferation and tubulogenesis, similar to effects seen when the cells were transfected with PKD siRNA (small interfering RNA). CRT5, a novel specific PKD inhibitor, will greatly facilitate the study of the role of PKD signalling mechanisms in angiogenesis.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/enzimologia , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação , Especificidade por Substrato , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
20.
Traffic ; 11(1): 161-74, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19883397

RESUMO

Vascular endothelial growth factor A (VEGF-A)-induced signaling through VEGF receptor 2 (VEGFR2) regulates both physiological and pathological angiogenesis in mammals. However, the temporal and spatial mechanism underlying VEGFR2-mediated intracellular signaling is not clear. Here, we define a pathway for VEGFR2 trafficking and proteolysis that regulates VEGF-A-stimulated signaling and endothelial cell migration. Ligand-stimulated VEGFR2 activation and ubiquitination preceded proteolysis and cytoplasmic domain removal associated with endosomes. A soluble VEGFR2 cytoplasmic domain fragment displayed tyrosine phosphorylation and activation of downstream intracellular signaling. Perturbation of endocytosis by the depletion of either clathrin heavy chain or an ESCRT-0 subunit caused differential effects on ligand-stimulated VEGFR2 proteolysis and signaling. This novel VEGFR2 proteolysis was blocked by the inhibitors of 26S proteasome activity. Inhibition of proteasome activity prolonged VEGF-A-induced intracellular signaling to c-Akt and endothelial nitric oxide synthase (eNOS). VEGF-A-stimulated endothelial cell migration was dependent on VEGFR2 and VEGFR tyrosine kinase activity. Inhibition of proteasome activity in this assay stimulated VEGF-A-mediated endothelial cell migration. VEGFR2 endocytosis, ubiquitination and proteolysis could also be stimulated by a protein kinase C-dependent pathway. Thus, removal of the VEGFR2 carboxyl terminus linked to phosphorylation, ubiquitination and trafficking is necessary for VEGF-stimulated endothelial signaling and cell migration.


Assuntos
Células Endoteliais/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/enzimologia , Citoplasma/metabolismo , Eletroforese em Gel de Poliacrilamida , Endossomos/efeitos dos fármacos , Endossomos/enzimologia , Endossomos/metabolismo , Células Endoteliais/enzimologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Humanos , Ligantes , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Lisossomos/metabolismo , Microscopia de Fluorescência , Neovascularização Patológica/enzimologia , Neovascularização Patológica/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Transporte Proteico
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