RESUMO
In this review we summarize the current understanding of signal transduction downstream of vascular endothelial growth factor A (VEGFA) and its receptor VEGFR2, and the relationship between these signal transduction pathways and the hallmark responses of VEGFA, angiogenesis and vascular permeability. These physiological responses involve a number of effectors, including extracellular signal-regulated kinases (ERKs), Src, phosphoinositide 3 kinase (PI3K)/Akt, focal adhesion kinase (FAK), Rho family GTPases, endothelial NO and p38 mitogen-activated protein kinase (MAPK). Several of these factors are involved in the regulation of both angiogenesis and vascular permeability. Tumour angiogenesis primarily relies on VEGFA-driven responses, which to a large extent result in a dysfunctional vasculature. The reason for this remains unclear, although it appears that certain aspects of the VEGFA-stimulated angiogenic milieu (high level of microvascular density and permeability) promote tumour expansion. The high degree of redundancy and complexity of VEGFA-driven tumour angiogenesis may explain why tumours commonly develop resistance to anti-angiogenic therapy targeting VEGFA signal transduction.
Assuntos
Neoplasias/irrigação sanguínea , Neovascularização Patológica/metabolismo , Transdução de Sinais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Permeabilidade Capilar , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
The complementary DNAs for wildtype and tyrosine kinase-inactivated (K634A) forms of the PDGF beta-receptor were expressed in porcine aortic endothelial cells. We examined the internalization and degradation of ligands and receptors after exposure of receptor expressing cells to PDGF-BB, which binds to the beta-receptor with high affinity, and PDGF-AB, which binds with lower affinity. Cells expressing wildtype beta-receptors were able to internalize and degrade the receptor, as well as the ligand, after exposure to PDGF-BB or -AB. Cells expressing the kinase-inactivated mutant receptor also internalized and degraded both receptor and ligand, but with lower efficiency compared with the wildtype receptor cells. The degradation of either form of receptor was inhibited by treatment of the cells with the lysosomotropic drug chloroquine. Exposure of wildtype and K634A receptor expressing cells to PDGF-AB resulted in a twofold slower rate of internalization of this ligand as compared with PDGF-BB, whereas the relative rate of degradation was similar for the two ligands. Our data indicate that tyrosine kinase activity promotes, but is not a prerequisite for, ligand-induced internalization and degradation of the ligand-receptor complex.
Assuntos
Endocitose , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Linhagem Celular , Cloroquina/farmacologia , Regulação para Baixo , Endotélio Vascular , Fibroblastos/metabolismo , Imunofluorescência , Humanos , Cinética , Proteínas Quinases/genética , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/genética , Receptores do Fator de Crescimento Derivado de Plaquetas , Pele , Suínos , TransfecçãoRESUMO
L6J1 rat myoblasts and rat skeletal muscle were studied for expression of mRNAs encoding PDGF A-chain, PDGF B-chain, PDGF alpha-receptor, and PDGF beta-receptor during in vitro and in vivo myoblast differentiation. RNA blot hybridizations demonstrated expression of the PDGF A-chain gene and the PDGF beta-receptor gene in L6J1 myoblasts and in crude muscle tissue isolated from developing rats. Transcripts of the PDGF A-chain were identified at all examined stages of in vitro and in vivo myogenic differentiation. Expression of the PDGF beta-receptor gene decreased in differentiated myotubes of L6J1 cells and in rat adult muscle tissue. Receptor binding assays demonstrated specific binding of PDGF-BB, but not -AA, to exponentially proliferating L6J1 myoblasts and to terminally differentiated L6J1 myotubes. The binding per cell nucleus was higher in exponentially proliferating myoblasts than in differentiated L6J1 myotubes. In serum free medium PDGF-BB was shown to increase c-fos protooncogene immunoreactivity in L6J1 myoblasts. In the presence of 0.5% FCS, PDGF-BB increased DNA synthesis in L6J1 myoblasts, while PDGF-AA showed no such effect. Differentiation, as monitored by myotube formation, was reduced in PDGF-BB-treated cultures. The possible role of PDGF in myoblast proliferation and differentiation is discussed.
Assuntos
Músculos/citologia , Fator de Crescimento Derivado de Plaquetas/genética , Receptores de Superfície Celular/genética , Animais , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular , Replicação do DNA/fisiologia , Imunofluorescência , Regulação da Expressão Gênica/fisiologia , Músculos/metabolismo , Fator de Crescimento Derivado de Plaquetas/biossíntese , Poli A/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-fos , RNA Mensageiro/metabolismo , Ratos , Receptores de Superfície Celular/biossíntese , Receptores do Fator de Crescimento Derivado de PlaquetasRESUMO
The expression of platelet-derived growth factor (PDGF) receptors in porcine uterus and human skin in situ, was compared with that of cultured primary cells isolated from the same tissues. PDGF receptor expression was examined by monoclonal antibodies specific for the B type PDGF receptor and by RNA/RNA in situ hybridization with a probe constructed from a cDNA clone encoding the B type PDGF receptor. In porcine uterus tissue both mRNA and the protein product for the PDGF receptor were detected in the endometrium; the myometrium, in contrast, contained much lower amounts. Moreover, freshly isolated myometrial cells were devoid of PDGF receptors. However, after 1 d in culture receptors appeared, and after 2 wk of culturing essentially all of the myometrial cells stained positively with the anti-PDGF receptor antibodies and contained PDGF receptor mRNA. Similarly, B type PDGF receptors were not detected in normal human skin, but fibroblast-like cells from explant cultures of human skin possessed PDGF receptors. When determined by immunoblotting, porcine uterus myometrial membranes contained approximately 20% of the PDGF receptor antigen compared with the amount found in endometrial membranes. In addition, PDGF stimulated the phosphorylation of a 175-kD component, most likely representing autophosphorylation of the B type PDGF receptor in endometrial membranes, whereas only a marginal phosphorylation was seen in myometrial membranes. Taken together, these results demonstrate that PDGF receptor expression varies in normal tissues and that fibroblasts and smooth muscle cells do not uniformly express the receptor in situ. Furthermore, fibroblasts and smooth muscle cells that are released from tissues are induced to express PDGF receptors in response to cell culturing. The data suggest that, in addition to the availability of the ligand, PDGF-mediated cell growth in vivo is dependent on factors regulating expression of the receptor.
Assuntos
Fibroblastos/metabolismo , Músculo Liso/metabolismo , Receptores de Superfície Celular/biossíntese , Animais , Anticorpos Monoclonais , Células Cultivadas/metabolismo , Fibroblastos/citologia , Humanos , Immunoblotting , Imuno-Histoquímica , Músculo Liso/citologia , Hibridização de Ácido Nucleico , Fosforilação , Receptores do Fator de Crescimento Derivado de Plaquetas , SuínosRESUMO
BACKGROUND: There is substantial evidence that phosphoinositide 3-kinase (PI 3-kinase) is a critical component of signalling pathways used by the cell-surface receptors for a variety of mammalian growth factors and other hormones. The physiological product of this enzyme is a highly polar membrane lipid called phosphatidylinositol (3,4,5)-trisphosphate This lipid has been postulated to act as a second-messenger in cells but its putative targets are still unknown. RESULTS: A particular rearrangement of actin filaments, which results in membrane ruffling, is elicited by the activation of PDGF beta-receptors expressed in cultured porcine aortic endothelial cells. We have found that this consequence of PDGF beta-receptor activation is inhibited by three independent manipulations of PI 3-kinase activity: firstly, by the deletion of tyrosine residues in the PDGF beta-receptor to which PI 3-kinase binds; secondly, by the overexpression of a mutant 85 kD PI 3-kinase regulatory subunit to which the catalytic kinase subunit cannot bind; and thirdly, by the addition of the fungal metabolite wortmannin, which is a potent inhibitor of the catalytic activity of PI 3-kinase. CONCLUSIONS: These results argue strongly that phosphatidylinositol (3,4,5)-trisphosphate synthesis is required for growth-factor-stimulated membrane ruffling in porcine aortic endothelial cells, and suggest that synthesis of this lipid may be part of a signalling pathway leading to direct or indirect activation of the small GTP-binding protein Rac.
Assuntos
Membrana Celular/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Androstadienos/farmacologia , Animais , Sítios de Ligação/genética , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Ativação Enzimática , Modelos Biológicos , Mutação , Fosfatidilinositol 3-Quinases , Fosfatos de Fosfatidilinositol/biossíntese , Fosforilação , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Suínos , WortmaninaRESUMO
BACKGROUND: Agonist-stimulated phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine, generating the putative messenger phosphatidate (PA). Proposed functions for PA, and hence for PLD, include kinase activation, the regulation of small molecular weight GTP-binding proteins, actin polymerization and secretion. It has not been possible to define a physiological function for PLD activation as it is generally stimulated together with other signalling pathways, such as those involving phospholipases A2 and C, phosphatidylinositide (PI) 3-kinase and the p21(ras)/mitogen-activated protein (MAP) kinase cascade. RESULTS: We report that, in porcine aortic endothelial (PAE) cells, lysophosphatidic acid (LPA) stimulated PLD activity and rapidly generated PA in the absence of other phospholipase, PI 3-kinase or MAP kinase activities. PLD activation was controlled by a tyrosine kinase-regulated pathway. LPA also stimulated actin stress fibre formation, but was inhibited by butan-1-ol; the alcohol also reduced the accumulation of PA. The addition of PA to cells did not stimulate PLD activity, but did cause stress fibre formation in a manner that was insensitive to butan-1-ol. Stimulation of stress fibre formation by LPA and PA was sensitive to genistein, and was inhibited by micro-injection of the Rho-inhibiting C3 exotoxin into PAE cells. CONCLUSIONS: This study provides the first clear demonstration of a physiological role for PLD activity. In PAE cells, the stimulation of actin stress fibre formation was a consequence of PA generation and, therefore, PLD activation. The results suggest that PA generation is upstream of Rho activation, and imply a role for PLD in the regulation of Rho-mediated pathways.
Assuntos
Actinas/metabolismo , Fosfolipase D/metabolismo , Animais , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Ativação Enzimática , Proteínas de Ligação ao GTP/metabolismo , Lisofosfolipídeos/farmacologia , Transdução de Sinais , SuínosRESUMO
BACKGROUND: Phosphoinositide 3-kinases (PI 3-kinases) are thought to play an important role in coordinating the responses elicited by a variety of growth factors, oncogene products and inflammatory stimuli. These responses include activation of membrane ruffling, chemotaxis, glucose transport, superoxide production, neurite outgrowth and pp70 S6 kinase. Some of these responses are also known to be regulated by Rac, a small GTP-binding protein related to Ras. Neither the transducing elements upstream of Rac, nor those downstream of PI 3-kinase, have been defined. RESULTS: We show here that platelet-derived growth factor (PDGF) can stimulate an increase in the level of GTP-Rac by at least two distinct mechanisms: firstly, by increased guanine nucleotide exchange; and secondly, by inhibition of a Rac GTPase activity. The first of these mechanisms is essential for the activation of Rac, and we show that it is dependent upon PDGR-stimulated synthesis of phosphatidylinositol (3,4,5)-trisphosphate. CONCLUSIONS: These results suggest that Rac activation lies downstream of PI 3-kinase activation on a PDGF-stimulated signalling pathway. Furthermore, as Rac has been implicated in at least two diverse cellular responses that are also though to require activation of PI 3-kinase--a reorganization of the actin cytoskeleton known as membrane ruffling and the neutrophil oxidative burst--these results suggest that Rac may be a major effector protein for the PI 3-kinase signalling pathway in many cell types.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular , Ativação Enzimática , Guanosina Trifosfato/metabolismo , Humanos , Fosfatidilinositol 3-Quinases , Proteínas rac de Ligação ao GTPRESUMO
A characteristic feature of the platelet-derived growth factor (PDGF) beta-receptor is the presence of an insert sequence in the protein tyrosine kinase domain. A receptor mutant which lacks the entire insert of 98 amino acids was expressed in CHO cells, and its functional characteristics were compared with those of the wild-type receptor. The mutant receptor bound PDGF-BB with high affinity and mediated internalization and degradation of the ligand with efficiency similar to that of the wild-type receptor but did not transduce a mitogenic signal. It was found to display a decreased autophosphorylation after ligand stimulation and had a decreased ability to phosphorylate exogenous substrates; phosphofructokinase was not phosphorylated at all, whereas a peptide substrate was phosphorylated, albeit at a lower rate compared with phosphorylation by the wild-type receptor. Furthermore, the mutant receptor did not mediate actin reorganization but mediated an increase in c-fos expression. The data indicate that the insert in the kinase domain of the PDGF beta-receptor is important for the substrate specificity or catalytic efficiency of the kinase; the deletion of the insert interferes with the transduction of some, but not all, of the signals that arise after activation of the receptor.
Assuntos
Deleção Cromossômica , Elementos de DNA Transponíveis , Genes , Proteínas Quinases/genética , Receptores de Superfície Celular/genética , Transdução de Sinais , Animais , Linhagem Celular , Clonagem Molecular , Expressão Gênica , Vetores Genéticos , Humanos , Cinética , Mutação , Fosforilação , Plasmídeos , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-fos , Proto-Oncogenes , Receptores do Fator de Crescimento Derivado de Plaquetas , TransfecçãoRESUMO
The structure of the human receptor for platelet-derived growth factor (PDGF) has been deduced through cDNA cloning. A 5.45-kilobase-pair cDNA clone predicts a 1,106-amino-acid polypeptide, including the cleavable signal sequence. The overall amino acid sequence similarity with the murine PDGF receptor is 85%. After transcription of the cDNA and translation in vitro, a PDGF receptor antiserum was used to immunoprecipitate a product of predicted size, which also could be phosphorylated in vitro. Stable introduction of the cDNA into Chinese hamster ovary (CHO) cells led to the expression of a 190-kilodalton component, which was immunoprecipitated by the PDGF receptor antiserum; this most probably represents the mature PDGF receptor. Binding assays with different 125I-labeled dimeric forms of PDGF A and B chains showed that the PDGF receptor expressed in CHO cells bound PDGF-BB and, to a lesser extent, PDGF-AB, but not PDGF-AA.
Assuntos
Clonagem Molecular , DNA/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptores de Superfície Celular/genética , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Genes , Humanos , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Biossíntese de Proteínas , Receptores de Superfície Celular/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , TransfecçãoRESUMO
Ligand stimulation of the platelet-derived growth factor (PDGF) beta-receptor leads to activation of its intrinsic tyrosine kinase and autophosphorylation of the intracellular part of the receptor. The autophosphorylated tyrosine residues mediate interactions with downstream signal transduction molecules and thereby initiate different signalling pathways. A pathway leading to activation of the GTP-binding protein Ras involves the adaptor molecule GRB2. Here we show that Tyr-716, a novel autophosphorylation site in the PDGF beta-receptor kinase insert, mediates direct binding of GRB2 in vitro and in vivo. In a panel of mutant PDGF beta-receptors, in which Tyr-716 and the previously known autophosphorylation sites were individually mutated, only PDGFR beta Y716F failed to bind GRB2. Furthermore, a synthetic phosphorylated peptide containing Tyr-716 bound GRB2, and this peptide specifically interrupted the interaction between GRB2 and the wild-type receptor. In addition, the Y716(P) peptide significantly decreased the amount of GTP bound to Ras in response to PDGF in permeabilized fibroblasts as well as in porcine aortic endothelial cells expressing transfected PDGF beta-receptors. The mutant PDGFR beta Y716F still mediated activation of mitogen-activated protein kinases and an increased DNA synthesis in response to PDGF, indicating that multiple signal transduction pathways transduce mitogenic signals from the activated PDGF beta-receptor.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Divisão Celular , Células Cultivadas , Análise Mutacional de DNA , Proteína Adaptadora GRB2 , Guanosina Trifosfato/metabolismo , Humanos , Ligantes , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Receptores Proteína Tirosina Quinases/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Análise de Sequência , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Two novel platelet-derived growth factor (PDGF) alpha-receptor transcripts of 1.5 kilobases and 5.0 kilobases are expressed in the human teratocarcinoma cell line Tera-2 only while the cells are in an undifferentiated state. After retinoic acid-induced differentiation, expression of these mRNAs is completely shut off and instead, the cells express a single 6.4-kilobase mRNA species which is also expressed in many other cell types. The 1.5-kilobase mRNA initiates within intron 12, contains the correctly spliced exons 13, 14, 15, and 16, and contains a cryptic exon, designated teratocarcinoma cryptic exon, at the 3' end. Teratocarcinoma cryptic exon contains a functional polyadenylation signal. Exons 13 to 16 correspond to the first tyrosine kinase domain and to part of the interkinase domain of the PDGF alpha-receptor. Recently, a splice variant lacking exon 14 was identified. These results show that a combination of alternative promoter usage and alternative splicing of the human PDGF alpha-receptor gene occur in a developmentally regulated fashion. In vitro translation of the 1.5-kilobase mRNA generates protein products which can be specifically immunoprecipitated with a PDGF alpha-receptor-specific antibody. The significance of the expression of this transcript for the growth factor-independent proliferation of undifferentiated Tera-2 cells is unclear. Expression of PDGF alpha-receptor transcripts containing the cryptic exon may be useful as a marker for undifferentiated stem cells in human teratocarcinomas.
Assuntos
Regulação Neoplásica da Expressão Gênica , Receptores do Fator de Crescimento Derivado de Plaquetas/análise , Teratocarcinoma/química , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , DNA de Neoplasias/análise , Éxons/genética , Humanos , Masculino , Dados de Sequência Molecular , RNA Mensageiro/análise , RNA Mensageiro/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Teratocarcinoma/patologia , Células Tumorais CultivadasRESUMO
The expression of platelet-derived growth factor (PDGF) and its receptors was analyzed in 14 gliomas of various degrees of malignancy and compared with three gliosis cases by in situ hybridization and immunohistochemistry techniques. Expression of both PDGF A- and B-chains was higher in glioblastomas than in astrocytomas. The PDGF A-chain mRNA was predominantly found in cell-rich areas in glioblastomas. The cognate PDGF-alpha receptor (PDGFR-alpha) mRNA was heterogeneously distributed in gliomas of all grades, and PDGFR-alpha expression was higher in gliomas than in gliosis. Within some glioblastomas probed with PDGFR-alpha complementary RNA, cells heavily loaded with grains were intermingled with others containing low or moderate signals. The heavily labeled cells were often found in the vicinity of proliferating capillaries. Immunostaining with an anti-PDGF antibody and an affinity-purified antiserum against the PDGFR-alpha showed strong staining of most tumor cells with both antibodies in glioblastoma. In addition, the PDGFR-alpha antibodies yielded a strong staining of scattered cells, and the anti-PDGF antibody yielded staining of a few cells within the astrocytoma. Furthermore, high levels of the PDGF-beta receptor (PDGFR-beta) and PDGF B-chain mRNA as well as the beta receptor protein were found in hyperplastic capillaries. These results suggest the presence of autocrine and paracrine loops in glioma, activating the PDGFR-alpha in glioma cells and the PDGFR-beta in endothelial cells.
Assuntos
Astrocitoma/patologia , Neoplasias Encefálicas/patologia , Glioma/patologia , Fator de Crescimento Derivado de Plaquetas/análise , RNA Mensageiro/análise , Receptores de Superfície Celular/análise , Adolescente , Adulto , Idoso , Astrocitoma/irrigação sanguínea , Astrocitoma/genética , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/genética , Capilares/patologia , Feminino , Glioma/genética , Humanos , Imuno-Histoquímica , Substâncias Macromoleculares , Masculino , Pessoa de Meia-Idade , Hibridização de Ácido Nucleico , Fator de Crescimento Derivado de Plaquetas/genética , RNA Mensageiro/genética , Receptores de Superfície Celular/genética , Receptores do Fator de Crescimento Derivado de PlaquetasRESUMO
Antithrombin is a plasma protein of the serpin superfamily that may occur as several conformational variants. The native form of antithrombin is a major regulator of blood clotting. In the present study, we have identified the mechanism underlying the antiangiogenic action of a heat-denatured form, denoted latent antithrombin. Fibroblast growth factor (FGF)-induced angiogenesis in the chick embryo and angiogenesis in mouse fibrosarcoma tumors were inhibited by treatment with latent antithrombin at 1 mg/kg/day. Thermolysin-cleaved and native antithrombin were less efficient in these respects. Treatment with latent antithrombin induced apoptosis of cultured endothelial cells and inhibited cell migration toward FGF-2. Under these conditions, FGF-2-stimulated FGF receptor kinase activity was unaffected. However, actin reorganization, activation of focal adhesion kinase, and focal adhesion formation were disturbed by latent antithrombin treatment of FGF-2-stimulated endothelial cells. These data indicate that latent antithrombin induces apoptosis of endothelial cells by disrupting cell-matrix interactions through uncoupling of focal adhesion kinase.
Assuntos
Inibidores da Angiogênese/farmacologia , Antitrombinas/farmacologia , Apoptose/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Fibrossarcoma/irrigação sanguínea , Neovascularização Patológica/tratamento farmacológico , Alantoide/irrigação sanguínea , Alantoide/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Comunicação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Inibição de Migração Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Embrião de Galinha , Córion/irrigação sanguínea , Córion/efeitos dos fármacos , Fatores de Crescimento Endotelial/antagonistas & inibidores , Endotélio Vascular/citologia , Matriz Extracelular/efeitos dos fármacos , Feminino , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Fibrossarcoma/tratamento farmacológico , Fibrossarcoma/patologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Linfocinas/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/patologia , Proteínas Tirosina Quinases , Receptores Proteína Tirosina Quinases , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Formal proof for an involvement of autocrine stimulation in the disturbed growth of malignant cells has been difficult to obtain, in part due to lack of precise methods of assessing growth factor production and receptor occurrence. In this study we have analyzed the mRNA levels for two growth factors and the corresponding receptors in a number of established human malignant glioma cell lines. Twenty-one tested lines all contained transcripts for the platelet-derived growth factor (PDGF) A chain while 16-17 of 21 expressed the c-sis/PDGF B chain gene; these two genes were expressed independently of each other. PDGF receptor transcripts were present in 15-16 of the 21 lines. Transcripts for the epidermal growth factor receptor were found in all 15 tested lines, in 2 of them at high levels, and the corresponding ligand transforming growth factor-alpha was found in 11 of 15 lines. No amplification or structural rearrangements of the genes, as analyzed by Southern blot hybridization, could explain the varying expression of PDGF A and B chain transcripts or the elevated levels of epidermal growth factor receptor mRNA. A correlation was found between cell morphology and expression of growth factor and receptor mRNA in these lines. The highest amount of PDGF receptor transcripts was found in cells with fibroblast-like morphology, and c-sis/B chain transcripts were found in small cell types and in cells with astrocyte-like morphology, while no clear relationship was found between PDGF receptor and A chain transcript levels or between morphology and A chain transcripts. It is possible that the findings reflect a coordinated expression of these genes in the progenitor cells. In conclusion, the data imply the existence of two possible autocrine loops in human malignant glioma lines, affecting the PDGF and epidermal growth factor receptor pathways.
Assuntos
Receptores ErbB/genética , Glioma/genética , Peptídeos/genética , Fator de Crescimento Derivado de Plaquetas/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/genética , Linhagem Celular , DNA/análise , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Glioma/metabolismo , Humanos , Poli A/análise , RNA/análise , Receptores do Fator de Crescimento Derivado de Plaquetas , Fatores de Crescimento TransformadoresRESUMO
We have examined fibroblast growth factor (FGF) receptor-1 mediated signal transduction in differentiation of endothelial cells (EC). The activated FGFR-1 couples to Ras through two adaptor proteins, FRS2 and Shc. In FGF-2 treated proliferating EC, FRS2 as well as Shc are tyrosine phosphorylated and interact with Grb2. In contrast, in FGF-2 treated differentiating cells, Shc, but not FRS2, is engaged in Grb2-interactions. Sustained MAP kinase activity has previously been implicated in differentiation. In FGF stimulated proliferating and differentiating endothelial cells, the MAP kinase Erk2 is activated in a sustained manner. Inhibition of MEK and MAP kinase activity by PD98059 treatment of cells, still allows EC tube formation. The FGFR-1 mediates activation of protein kinase C (PKC) through direct binding and activation of phospholipase C-gamma (PLC-gamma), and has also been shown to activate the cytoplasmic tyrosine kinase Src. Treatment of the cells with the PKC inhibitor bisindolylmaleimide does not prevent tube formation. In contrast, Src kinase activity is a prerequisite for EC differentiation, since treatment of the cells with PP1, a Src family specific inhibitor, abrogates tube formation. In differentiating EC, FGF-2 induces complex formation between Src and focal adhesion kinase (FAK). These data indicate that the Ras pathway is initiated via Shc or FRS2, dependent on the cellular program. Blocking the function of Src family kinases, attenuates differentiation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Endotélio Vascular/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Genes ras , Domínios de Homologia de src , Animais , Becaplermina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/efeitos dos fármacos , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Moléculas de Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Flavonoides/farmacologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Proteína Adaptadora GRB2 , Indóis/farmacologia , Isoenzimas/efeitos dos fármacos , Isoenzimas/metabolismo , Maleimidas/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Fosfolipase C gama , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/efeitos dos fármacos , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-sis , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/efeitos dos fármacos , Fosfolipases Tipo C/metabolismo , Proteínas ras/genéticaRESUMO
Fibroblast growth factors (FGFs) induce proliferation and differentiation of a wide variety of cells by stimulation of cell surface expressed high affinity-binding receptor tyrosine kinases. Members of the Src family of cytoplasmic tyrosine kinases are substrates for certain growth factor receptors. We have examined interactions between FGF receptor-1 (FGFR-1) and Src, Fyn and Yes. In lung capillary endothelial cells and murine fibroblasts, bFGF stimulation led to increased autophosphorylation of Src family members. In contrast, in porcine aortic endothelial cells (FGFR-1/PAE) and lung fibroblasts from chinese hamster (CCL 39), activation of FGFR caused reduced autophosphorylation of Src and Fyn. In neither case could complex-formation between Src members and FGFR be seen. Analysis of a panel of mutated FGFR-1 expressed in PAE cells showed that FGFR-1/Y766F mediated an increased autophosphorylation of Src members and upregulation of their kinase activities. Y766 in FGFR-1 has been shown to serve as a binding site for phospholipase C-gamma, which regulates Ca2+ fluxes and protein kinase C (PKC) activity. The negative effect on Src kinase activity upon FGFR stimulation was mimicked by activation of PKC in FGFR-1/PAE or CCL 39 cells using phorbol 12-myristate 13-acetate (PMA) and Src was phosphorylated in vitro by purified recombinant PKC alpha. Moreover, inhibition of PKC attenuated the bFGF induced decrease in autophosphorylation of Src family members. These data indicate a negative regulatory role for PKC on Src kinase activity in certain cell types.
Assuntos
Proteína Oncogênica pp60(v-src)/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Animais , Linhagem Celular , Regulação para Baixo , Fosforilação , Proteína Quinase C/fisiologia , SuínosRESUMO
Vascular endothelial growth factor (VEGF) and placenta growth factor (PIGF) are structurally related growth factors for endothelial cells. VEGF binds to the related receptor tyrosine kinases Flt 1 and KDR/Flk 1 with high affinity, whereas PlGF binds only to Flt 1. Ligand-stimulated KDR is known to transduce signals for cellular activity such as proliferation and migration, whereas weak or no responses have been recorded for Flt 1. We examined VEGF and PlGF for their capacity to stimulate signal transduction in porcine aortic endothelial cells expressing Flt 1 or KDR. VEGF had essentially no effect on Flt 1 expressing cells, but induced DNA synthesis and migration of KDR expressing cells. PIGF on the other hand induced DNA synthesis but not migration of the Flt 1 cells. In agreement, MAP kinase, examined as a marker for DNA synthesis, was activated both by VEGF-stimulation of the KDR cells and by PlGF-stimulation of the Flt 1 cells. In contrast, phospholipase C-gamma (PLC-gamma), was tyrosine phosphorylated only in VEGF stimulated KDR cells, and not in the PlGF-stimulated Flt 1 cells, which is in agreement with a role for PLC-gamma in cellular migration. We furthermore examined induction of protein levels of plasminogen activator (PA), which was evident in the PlGF-stimulated Flt 1 cells, but not in the VEGF-stimulated KDR cells. These data show that Flt 1 is able to mediate an array of biological signals when appropriately stimulated and that the pattern of responses of PlGF-stimulation of Flt 1 is distinct from the pattern of responses to VEGF-stimulation of KDR.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Endotélio Vascular/metabolismo , Isoenzimas/metabolismo , Mitógenos/farmacologia , Proteínas da Gravidez/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Aorta/citologia , Movimento Celular , Células Cultivadas , DNA/biossíntese , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Expressão Gênica , Humanos , Linfocinas/farmacologia , Fosfolipase C gama , Fosforilação , Fator de Crescimento Placentário , Ativadores de Plasminogênio/biossíntese , Ativadores de Plasminogênio/metabolismo , Proteínas Proto-Oncogênicas/biossíntese , Receptores Proteína Tirosina Quinases/biossíntese , Receptores de Fatores de Crescimento/biossíntese , Receptores de Fatores de Crescimento do Endotélio Vascular , Suínos , Tirosina/metabolismo , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Signal transducers and activators of transcription (Stats) are known to transduce signals from the cell surface to the nucleus in cytokine receptor signaling. We examined the capacity of platelet-derived growth factor (PDGF) receptor to interact with and activate Stat molecules. Activation of the PDGF beta-receptor led to tyrosine phosphorylation of Stat1, Stat3 and Stat5, which was accompanied by specific DNA-binding activities. These events were only weakly stimulated by the activated PDGF alpha-receptor. In cells expressing PDGF beta-receptors mutated at Tyr579, Tyr581 or Tyr775, tyrosine phosphorylation as well as DNA-binding activity of Stat5 was reduced. Immobilized peptides containing phosphorylated Tyr579, Tyr581 or Tyr775 bound Stat5, suggesting direct binding of Stat5 to these tyrosine residues of the PDGF beta-receptor. Members of the Janus kinase family were also shown to interact with the PDGF beta-receptor, and to a lesser extent with the alpha-receptor, but their importance for PDGF-induced Stat activation remains to be determined.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas do Leite , Fator de Crescimento Derivado de Plaquetas/fisiologia , Proteínas Tirosina Quinases/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Células 3T3 , Animais , Células COS , DNA/metabolismo , Janus Quinase 3 , Camundongos , Fosforilação , Receptor alfa de Fator de Crescimento Derivado de Plaquetas , Receptor beta de Fator de Crescimento Derivado de Plaquetas , Fator de Transcrição STAT1 , Fator de Transcrição STAT2 , Fator de Transcrição STAT3 , Fator de Transcrição STAT4 , Fator de Transcrição STAT5RESUMO
Endothelial cells expressing fibroblast growth factor receptor-1 (FGFR-1) migrate and proliferate in response to treatment with FGF. We analysed ligand-induced migration and proliferation of porcine aortic endothelial cells expressing wild-type FGFR-1, point-mutated Y766F FGFR-1, unable to activate phospholipase C-gamma1 (PLC-gamma1), or carboxyl-terminally truncated FGFR-1, lacking either 48 (from amino acid 774 in the FGFR-1 sequence) or 63 (from amino acid 759) amino acid residues of the C-terminal tail. The truncated CT63 FGFR-1 mutant failed to mediate chemotaxis, but was in response to ligand stimulation capable of mediating proliferation of the cells, stimulation of MAP kinase activity and tyrosine phosphorylation of FRS2, an FGFR-1 specific signaling molecule. The defect in migration-capacity of CT63 was not due to loss of Y766, and thereby PLC-gamma1 activation, since cells expressing the mutant Y766F FGFR-1 migrated as efficiently as the wild-type receptor cells. Induction of phospholipase A2 (PLA2) activity by the activated FGFR-1 was dependent on the presence of Y766, and was therefore also not critical for the chemotactic response. Although the FGFR-1 only very inefficiently mediates activation of phosphatidylinositol 3' kinase (PI 3-kinase), the PI 3-kinase inhibitor wortmannin suppressed wild-type FGFR-1 mediated migration. We conclude that the signal transduction pathway for FGFR-1 mediated migration is independent of phosphotyrosine residues in the receptor and requires activation of a wortmannin-sensitive enzyme.