RESUMO
BACKGROUND AND PURPOSE: P2Y(1) is a purine receptor that triggers platelet aggregation. Its inhibition was studied in patients with stable coronary artery disease (CAD) receiving standard anti-platelet therapy. EXPERIMENTAL APPROACH: Blood samples from 10 patients on aspirin therapy (ASA, 80 mg·day(-1) ) were withdrawn before and 24 h after the administration of 450 mg clopidogrel (ASA/C) and were anti-coagulated with citrate or hirudin/PPACK in the presence or absence of the P2Y(1 ) inhibitor MRS2179 (M, 100 µM). Platelet responses to ADP (2.5 µM) and TRAP (2.5 µM), and collagen-induced thrombosis under flow conditions were analysed. KEY RESULTS: Compared with ASA, ASA + M strongly inhibited ADP-induced peak platelet aggregation (88%), late aggregation (84%), P-selectin expression (85%) and α(IIb) ß(3) activation (62%) (28%, 65%, 70% and 51% inhibition, respectively, for ASA/C vs. ASA). ASA + M also inhibited platelet/monocyte and platelet/neutrophil conjugate formation by 69% and 71% (57% and 59% for ASA/C vs. ASA). In TRAP-activated blood, ASA + M unexpectedly inhibited α(IIb) b(3) activation by 30%. In blood perfused in collagen-coated glass capillaries (shear rate of 1500 s(-1) ), ASA/C prevented thrombus growth beyond 5 min in relation to thrombus fragments embolization. ASA + M with or without clopidogrel completely prevented thrombus formation. Finally, ex vivo addition of MRS2179 and ASA to the blood of healthy donors markedly blocked thrombus formation on collagen in flow conditions, in contrast to ASA plus the P2Y(12) inhibitor 2-MeSAMP. CONCLUSIONS AND IMPLICATIONS: Through particularly efficient complementarities with ASA to inhibit platelet activation and thrombus formation, the inhibition of P2Y(1) in the blood of patients with CAD appears to play a more important role than previously anticipated.
Assuntos
Aspirina/farmacologia , Doença da Artéria Coronariana/tratamento farmacológico , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Ticlopidina/análogos & derivados , Difosfato de Adenosina/administração & dosagem , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/farmacologia , Idoso , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Clopidogrel , Colágeno/administração & dosagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores da Agregação Plaquetária/farmacologia , Receptores Purinérgicos P2Y1/efeitos dos fármacos , Receptores de Trombina/administração & dosagem , Trombose/prevenção & controle , Ticlopidina/farmacologiaRESUMO
The mitogen-activated protein (MAP) kinases ERK2, p38 and JNK1 are present in platelets and are activated by various stimuli, such as thrombin, collagen, von Willebrand factor (VWF) and ADP. Until recently, MAP kinases were only studied in the conventional model of agonist-induced platelet aggregation mediated by fibrinogen and integrin alphaIIbbeta3. However, this approach is likely to be too limited for a physiological understanding of platelet MAP kinases and their signaling pathways. Recent studies with varying blood-flow conditions and animal models of thrombosis have provided deeper insight into the role of MAP kinases in thrombus formation and the dependence of these kinases on shear conditions. This review summarizes and discusses the physiological functions of these kinases in hemostasis and thrombosis as revealed by various technical approaches.
Assuntos
Hemostasia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Trombose , Animais , Plaquetas/enzimologia , HumanosRESUMO
BACKGROUND: Protease nexin-1 (PN-1) is an important physiological regulator of thrombin in the brain. PN-1 is also present in aortic smooth muscle cells and may thus participate in vascular biology. However, little is known about its function in the vessel wall. OBJECTIVES: In this study, we investigated the effect of PN-1 overexpression in smooth muscle cells (SMCs), on their sensitivity to thrombin, and their capacity for adhesion, spreading and migration. RESULTS: Two clones exhibiting a two- to threefold increase in PN-1 expression were selected and compared with untransfected and mock-transfected cells. Overexpression of PN-1 was observed to inhibit thrombin-induced cell responses as indicated by a twofold decrease in induction of PAI-1 expression, a decreased calcium mobilization in response to low thrombin concentrations and a twofold increase in the capacity to inhibit thrombin catalytic activity. Overexpression of PN-1 did not modify adhesion, spreading, and migration of SMCs on type I collagen. In contrast, SMCs overexpressing PN-1 exhibited a 40% reduction in adhesion, a 50% reduction in spreading and a complete absence of migration on vitronectin when compared with control SMCs. CONCLUSIONS: Our studies thus reveal that PN-1 is likely to play a critical role in regulating essential cell functions such as (i) thrombin-induced responses, which are dependent on its antiprotease activity, and (ii) adhesion, spreading, and migration, which are independent of its antiprotease activity and may be related to its interaction with other partners, such as vitronectin in the present case.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Músculo Liso Vascular/metabolismo , Receptores de Superfície Celular/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Sinalização do Cálcio/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , DNA Complementar/genética , Expressão Gênica , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Nexinas de Proteases , Ratos , Receptores de Superfície Celular/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trombina/farmacologia , Transfecção , Vitronectina/metabolismoRESUMO
The functional significance of extracellular signal-regulated kinase 2 (ERK2) activation was investigated during shear induced human platelet aggregation (SIPA) in vitro and during shear controlled thrombosis in vivo in intestinal arterioles and venules of wild type (WT) and transgenic (TG) mice with platelet-specific overexpression of human P2X(1) (TG). In SIPA, ERK2 was rapidly phosphorylated during GPIb stimulation, its activation contributing to SIPA for 50%, independently of P2X(1) regulation. Thrombotic occlusion of injured arterioles occurred considerably faster in TG (4.3 +/- 2.3 min) than in WT (38 +/- 8 min) arterioles, but occlusion times in TG (19 +/- 12) and WT (48 +/- 4.5 min) venules differed less. Both the alphabeta-meATP triggered desensitization of platelet P2X(1), as well as P2X(1) antagonism by NF279 or NF449 prolonged mean occlusion to about 75 min in WT and 65 min in TG arterioles, but venular occlusion times were less affected. Preventing ERK2 activation by U0126 prolonged occlusion times in TG (41 +/- 10 min) and WT (51 +/- 17) arterioles more than in TG (46 +/- 5 min) and WT (56 +/- 6 min) venules, uncovering a role for ERK2 in shear controlled thrombosis. Antagonism of GPIb by a recombinant murine von Willebrand factor (VWF)-A1 fragment prolonged occlusion times to comparable values, ranging from 55 to 58 min, both in TG and WT arterioles and venules. Further inhibition strategies, combining VWF-A1, U0126 and NF449 in WT and TG mice and resulting in occlusion in various time windows, identified that inhibition by VWF-A1 largely abrogated the ERK2 contribution to thrombosis. In conclusion, P2X(1) and ERK2 both participate in shear stress controlled thrombosis, but ERK2 activation is initiated predominantly via GPIb-VWF interactions.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/sangue , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Receptores Purinérgicos P2/sangue , Trombose/sangue , Trombose/enzimologia , Animais , Arteríolas , Ativação Enzimática , Humanos , Técnicas In Vitro , Camundongos , Camundongos Transgênicos , Fosforilação , Agregação Plaquetária , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X , Proteínas Recombinantes/sangue , Proteínas Recombinantes/genética , Estresse Mecânico , Trombose/etiologia , Trombose Venosa/sangue , Trombose Venosa/enzimologia , Trombose Venosa/etiologia , Fator de von Willebrand/metabolismoRESUMO
The adhesion of cells to the extracellular matrix plays a major role in cell migration. Pretreatment with platelet-derived growth factor (PDGF) inhibited the adhesion of smooth muscle cells to fibronectin by 80%. This inhibition decreased as concentrations of fibronectin increased. In the presence of 200 microm GRGDS peptide, only 45% of PDGF-treated cells adhered to fibronectin compared with 80% of control cells. This indicates that a decrease in integrin avidity was induced by PDGF. Cell adhesion was partially restored when the activation of the extracellular signal-regulated kinase (ERK) was inhibited with PD98059. The remaining inhibition of adhesion (50%) was independent of the fibronectin concentration, suggesting that the ERK pathway is involved in the decrease in integrin avidity. This was confirmed by depleting ERK protein levels by treatment with ERK antisense oligonucleotide. The adhesion of ERK control oligonucleotide-treated cells decreased by 41% when the concentration of GRGDS peptide was increased from 50 to 200 microm but only decreased by 11% in ERK antisense oligonucleotide-treated cells. Treatment with PDGF also delayed focal complex assembly and inhibited stress fiber formation. Consistent with a delay in tyrosine phosphorylation of paxillin, PDGF treatment caused a lag in focal complex formation, although this was not associated with any change in Src family tyrosine kinase activity. Our results indicate that PDGF inhibits smooth muscle cells adhesion by two pathways. The first involves an ERK-dependent decrease in integrin avidity; the second involves the ERK-independent inhibition of focal complex assembly.
Assuntos
Fibronectinas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso/citologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Aorta/metabolismo , Adesão Celular , Movimento Celular , Proteínas do Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Matriz Extracelular/metabolismo , Flavonoides/farmacologia , Immunoblotting , Sistema de Sinalização das MAP Quinases , Microscopia de Fluorescência , Oligonucleotídeos Antissenso/metabolismo , Oligopeptídeos/farmacologia , Paxilina , Peptídeos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Inibidores da Agregação Plaquetária/farmacologia , Testes de Precipitina , Suínos , Fatores de Tempo , Tirosina/metabolismo , Quinases da Família src/metabolismoRESUMO
Fibroblast growth factor 1 (FGF1) induces proliferation and differentiation in a wide variety of cells of mesodermal and neuroectodermal origin. FGF1 has no 'classical' signal sequence to direct its secretion, and there has been considerable debate concerning FGF1 secretion and its role in the biological activities of FGF1. We investigated the effects of FGF1 secretion and the signalling induced by signal peptide (SP)-containing FGFI and SP-less FGF1, on the proliferation and the apoptosis in retinal pigmented epithelial (RPE) cells. Primary RPE cell cultures were transfected with FGF1 (FGF1 cells) and SP-FGF1 (SP-FGF1 cells) cDNAs. SP-FGF1 cells secreted large amount of FGF1 and actively proliferated, whereas FGF1 and control cells did not. Secreted FGF1 induced short-term activation of both FGFR1 and ERK2, which were required for cell proliferation. In contrast, SP-FGF1 cells stopped secreting FGF1 and died rapidly, if cultured in the absence of serum. Surprisingly, FGF1 cells, but not control cells, secreted FGF1 and were resistant to apoptosis induced by serum depletion. Secreted FGF1 induced long-term activation of FGFR1 and ERK2, which was necessary to induce a constant and high level of Bcl-x production, and to induce cell survival in FGFI cells. Downregulation of ERK2 and Bcl-x increased apoptosis. Thus, the proliferation and survival activities of FGF1 depend on the secretion of FGF1 which is determined by the cell culture conditions. Cell proliferation was SP-dependent, whereas cell survival was not. The signal peptide controls the level and duration, 'whispering or shouting', of ERK2 activation cells which determines FGF1 biological function and may have important implications for anti-degenerative and anti-proliferative treatments.
Assuntos
Proteínas do Olho/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Epitélio Pigmentado Ocular/citologia , Animais , Apoptose/efeitos dos fármacos , Bovinos , Divisão Celular , Sobrevivência Celular , Células Cultivadas , Meios de Cultura Livres de Soro/farmacologia , DNA Complementar/genética , Ativação Enzimática , Fator 1 de Crescimento de Fibroblastos , Fator 2 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosforilação , Epitélio Pigmentado Ocular/metabolismo , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/biossíntese , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Transdução de Sinais , Transfecção , Proteína bcl-XRESUMO
Retinal pigmented epithelial (RPE) cells are of central importance in the maintenance of neural retinal function. Changes in the RPE cells associated with repair activities have been described as metaplasia, while RPE cell apoptosis is responsible for the development of a variety of retinal degenerations. We investigated the regulation of the anti-apoptotic properties of the fibroblast growth factors (FGF) 2 in serum-free cultures of RPE cells. In the absence of serum, confluent stationary RPE cells died by apoptosis via a caspase 3-dependent pathway. The addition of FGF2 greatly reduced apoptosis over a 7-day culture period. We demonstrated the involvement of an autocrine loop involving endogenous FGF1 in the mechanisms that govern FGF2-induced resistance to apoptosis by showing: (1) higher levels of apoptosis in cells treated with antisense FGF1 oligonucleotide or after neutralization of excreted FGF1; (2) the long-term activation of FGFR1 and of ERK2, (3) the inhibition of FGFR1 and ERK2 activation and an increase in apoptosis if excreted FGF1 was neutralized. FGF2 also increased the de novo synthesis and the production of Bcl-xl before the onset of apoptosis. Both inhibition of ERK2 activation, which decreased Bcl-xl synthesis, and downregulation of Bcl-x by antisense oligonucleotide treatment inhibited the survival-promoting activity of FGF2. Thus, FGF2-induced cell survival is a progressive adaptive phenomenon involving ERK2 activation by excreted FGF1 and ERK2-dependent Bcl-x production.
Assuntos
Apoptose/fisiologia , Fator 2 de Crescimento de Fibroblastos/biossíntese , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Epitélio Pigmentado Ocular/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Animais , Caspase 3 , Inibidores de Caspase , Caspases/metabolismo , Bovinos , Sobrevivência Celular , Células Cultivadas , Meios de Cultura Livres de Soro , Inibidores de Cisteína Proteinase/farmacologia , Fator 1 de Crescimento de Fibroblastos , Fator 2 de Crescimento de Fibroblastos/genética , Oligonucleotídeos Antissenso/farmacologia , Oligopeptídeos/farmacologia , Epitélio Pigmentado Ocular/efeitos dos fármacos , Epitélio Pigmentado Ocular/patologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Proteína bcl-XRESUMO
Platelets are an interesting model for studying the relationship betwen adhesion and mitogen-activated protein (MAP) kinase activation. We have recently shown that in platelets, ERK2 was activated by thrombin and downregulated by alpha(IIb)beta(3) integrin engagement. Here we focused our attention on the c-Jun NH2-terminal kinases (JNKs) and their activation in conditions of platelet aggregation. We found that JNK1 was present in human platelets and was activated after thrombin induction. JNK1 phosphorylation was detected with low concentrations of thrombin (0. 02 U/mL) and after 1 minute of thrombin-induced platelet aggregation. JNK1 activation was increased (fivefold) when fibrinogen binding to alpha(IIb)beta(3) integrin was inhibited by the Arg-Gly-Asp-Ser (RGDS) peptide or (Fab')(2) fragments of a monoclonal antibody specific for alpha(IIb)beta(3), demonstrating that, like ERK2, alpha(IIb)beta(3) integrin engagement negatively regulates JNK1 activation. Comparison of JNK1 activation by thrombin in stirred and unstirred platelets in the presence of RGDS peptide showed a positive regulation by stirring itself, independently of alpha(IIb)beta(3) integrin engagement, which was confirmed in a thrombasthenic patient lacking platelet alpha(IIb)beta(3). The same positive regulation by stirring was found for ERK2. These results suggest that MAP kinases (JNK1 and ERK2) are activated positively by thrombin and stirring. In conclusion, we found that JNK1 is present in platelets and can be activated after thrombin induction. Moreover, this is the first report showing that two different MAP kinases (ERK2 and JNK1) are regulated negatively by alpha(IIb)beta(3) engagement and positively by mechanical forces in platelets.
Assuntos
Plaquetas/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Ativação Plaquetária , Transdução de Sinais , Células Cultivadas , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Fosforilação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologiaRESUMO
Fibroblast growth factors (FGF) 1 and 2 and their tyrosine kinase receptor (FGFR) are present throughout the adult retina. FGFs are potential mitogens, but adult retinal cells are maintained in a nonproliferative state unless the retina is damaged. Our work aims to find a modulator of FGF signaling in normal and pathological retina. We identified and sequenced a truncated FGFR1 form from rat retina generated by the use of selective polyadenylation sites. This 70-kDa form of soluble extracellular FGFR1 (SR1) was distributed mainly localized in the inner nuclear layer of the retina, whereas the full-length FGFR1 form was detected in the retinal Muller glial cells. FGF2 and FGFR1 mRNA levels greatly increased in light-induced retinal degeneration. FGFR1 was detected in the radial fibers of activated retinal Muller glial cells. In contrast, SR1 mRNA synthesis followed a biphasic pattern of down- and up-regulation, and anti-SR1 staining was intense in retinal pigmented epithelial cells. The synthesis of SR1 and FGFR1 specifically and independently regulated in normal and degenerating retina suggests that changes in the proportion of various FGFR forms may control the bioavailability of FGFs and thus their potential as neurotrophic factors. This was demonstrated in vivo during retinal degeneration when recombinant SR1 inhibited the neurotrophic activity of exogenous FGF2 and increased damaging effects of light by inhibiting endogenous FGF. This study highlights the significance of the generation of SR1 in normal and pathological conditions.
Assuntos
Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Fator 2 de Crescimento de Fibroblastos/genética , Retina/fisiologia , Degeneração Retiniana/fisiopatologia , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Receptores ErbB/metabolismo , Éxons , Fator 1 de Crescimento de Fibroblastos , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Luz/efeitos adversos , Camundongos , Dados de Sequência Molecular , Epitélio Pigmentado Ocular/citologia , Epitélio Pigmentado Ocular/fisiologia , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos F344 , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Retina/citologia , Retina/fisiopatologia , Degeneração Retiniana/etiologia , Degeneração Retiniana/patologia , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica/efeitos da radiaçãoRESUMO
Retinal-pigmented epithelial (RPE) cell survival is critical to the maintenance of the function of the neural retinal and in the development of various retina degenerations. We investigated molecular mechanisms involved in this function by assessing apoptosis in RPE cells following serum deprivation. Apoptosis induced by serum withdrawal is lower in aged RPE cells because of higher endogenous acidic fibroblast growth factor (FGF1) synthesis and secretion. These experiments examined several aspects of FGF signaling and the contribution of endogenous FGF1 to activation of the extracellular signal-regulated kinase 2 (ERK2). In aged RPE cells, FGFR1 was rapidly activated, and its autophosphorylation followed the kinetics of endogenous FGF1 secretion, before the onset of apoptosis. ERK2 phosphorylation, activity, and de novo synthesis increased at the same time. In marked contrast, no de novo JNK1 synthesis was observed. MEK1 inhibition resulted in lower levels of ERK2 activation and synthesis and higher levels of apoptosis. Treatment with neutralizing anti-FGF1 or blocking anti-FGFR1 antibodies mimics these effects. Thus, this study strongly suggests that the survival-increasing effect of FGF1 in aged RPE cells is because of an autocrine/paracrine loop in which the ERK2 cascade plays a pivotal role.
Assuntos
Apoptose/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Epitélio Pigmentado Ocular/enzimologia , Animais , Bovinos , Células Cultivadas , Meios de Cultura Livres de Soro , Ativação Enzimática , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Fosforilação , Epitélio Pigmentado Ocular/citologia , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/fisiologia , Regulação para CimaRESUMO
Activation of the mitogen-activated protein (MAP) kinase pathway in nucleated cells is dependent on both growth factor receptors and integrins engaged in cell adhesion. Human platelets are an interesting model for studying cell adhesion and the involvement of integrin engagement on extracellular signal-regulated kinase (ERK) activation, independently from the nuclear-DNA signal pathway. Maximal phosphorylation and activity of ERK2 occurred late during thrombin-induced platelet aggregation (90 s and later), an alphaIIbbeta3 integrin-dependent event. Surprisingly, alphaIIbbeta3 inhibition by the RGDS ligand peptide, or (Fab')2 fragments of the AP-2 monoclonal antibody, resulted in a 2-fold enhancement in ERK2 phosphorylation and activity. A similar 2-fold enhancement of ERK2 activation was observed in thrombasthenic platelets which are defective in alphaIIbbeta3 and do not aggregate. This suggests that ERK2 activation in thrombin-induced platelet aggregation is dependent on thrombin rather than on alphaIIbbeta3 and is down-regulated by alphaIIbbeta3 engaged in ligand (fibrinogen) binding and/or aggregation. Finally, in the absence of stirring which allows fibrinogen binding to alphaIIbbeta3 but prevents aggregation, ERK2 was again overactivated. This overactivation appears to be consecutive to inhibition of aggregation itself and to alphaIIbbeta3 ligand binding. We conclude that in platelets, alphaIIbbeta3 engaged in aggregation down-regulates thrombin-induced ERK2 activation. To our knowledge, this is the first report of a down-regulation of the MAP kinase pathway by integrin engagement.
Assuntos
Plaquetas/enzimologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/farmacologia , Proteínas Tirosina Quinases/metabolismo , Ativação Enzimática , Humanos , Proteína Quinase 1 Ativada por Mitógeno , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Transdução de Sinais , Trombina/metabolismo , Trombina/farmacologiaRESUMO
U46619, a thromboxane A2 analogue, and basic fibroblast growth factor (FGF-2) both induced the expression of the inducible cyclo-oxygenase (Cox)-2 in porcine aortic smooth-muscle cells. This induction was dose-dependent (submaximal at 300 nM for U46619 and 1 ng/ml for FGF-2) and time-dependent, with similar intensity and maximal expression at 2 h. Under these conditions, both inducers stimulated rapid activation of extracellular signal-regulated kinase (ERK2) at 5-10 min, a transient and lower intensity being induced by U46619 whereas that induced by FGF-2 was sustained (>1 h). PD98059, an inhibitor of the ERK pathway, inhibited the expression of Cox-2. In contrast, activation of Jun-N-terminal kinase (JNK1) was sustained with U46619 but poorly induced by FGF-2. Cox-2 expression induced by U46619 or FGF-2 was similarly reduced by prostaglandin (PGE2), forskolin or dibutyryl-cAMP, suggesting a regulatory effect of adenylate cyclase on Cox-2 expression. However, activation of ERK2 by FGF-2 was not affected by PGE2 whereas that of JNK1 by U46619 was inhibited, suggesting that inhibition of COX-2 expression by cAMP may be downstream of ERK2. The effects of PGE2 and forskolin on Cox-2 and phosphorylation of JNK1 were reversed with the protein kinase A inhibitor H89. In addition, endogenous PGE2 down-regulated the expression of Cox-2 by the two inducers, as stimulation of the cells in the presence of different Cox inhibitors increased the expression of the protein. Overall, these results suggest that exogenous and endogenous PGE2 exert negative inhibitory effects on Cox-2 expression mediated by stimulation of protein kinase A.
Assuntos
Dinoprostona/farmacologia , Fator 2 de Crescimento de Fibroblastos/biossíntese , Isoenzimas/biossíntese , Músculo Liso Vascular/enzimologia , Endoperóxidos Sintéticos de Prostaglandinas/farmacologia , Prostaglandina-Endoperóxido Sintases/biossíntese , Tromboxano A2/análogos & derivados , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Animais , Aorta Torácica , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , AMP Cíclico/fisiologia , Ciclo-Oxigenase 2 , Dinoprostona/fisiologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Isoenzimas/efeitos dos fármacos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Suínos , Tromboxano A2/farmacologia , Fatores de Tempo , Vasoconstritores/farmacologiaRESUMO
The relationship between Rap1 proteins and cell proliferation was assessed by investigating the effect of isoforms AA and BB of platelet-derived growth factor (PDGF) on Rap1 protein and mRNA expression throughout the smooth muscle cell cycle. Firstly, PDGF BB-induced cell cycle traverse was studied, thus demonstrating entry into S phase at 18 to 20 h. Western blotting carried out on total Rap1 proteins showed that 5 ng/ml of PDGF BB instigated a biphasic induction of total Rap1 proteins during the cell cycle. This involved a 2.1 +/- 0.4-fold increase at 6 h (early G1) and a 2.8 +/- 0.6-fold increase at 20 to 24 h (G1/S transition). Such an up-regulation was abolished by addition of 1 ng/ml of transforming growth factor-beta 1 (TGF-beta 1), which inhibited up to 80% of the PDGF BB-induced entry into S phase. Comparative RT-PCR of both rap1a and rap1b mRNAs throughout the cell cycle allowed us to differentiate between the two rap1a and rap1b species. PDGF BB induced a 1.9 +/- 0.3-fold increase at 4 h and a 2.4 +/- 0.2-fold relative increase at 16 h for rap1b mRNA, whereas a unique 1.9 +/- 0.5-fold increase in rap1a mRNA was observed at 14 h. Again, this induction of rap1a and rap1b mRNAs by PDGF BB was totally abolished by TGF-beta 1. We conclude that the differential up-regulation of Rap1a and Rap1b proteins during the smooth muscle cell cycle is directly linked to cell proliferation.
Assuntos
Ciclo Celular/fisiologia , Proteínas de Ligação ao GTP/genética , Músculo Liso Vascular/química , Proteínas Proto-Oncogênicas/genética , Animais , Anticoagulantes/farmacologia , Aorta/citologia , Becaplermina , Southern Blotting , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Cinética , Músculo Liso Vascular/citologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-sis , RNA Mensageiro/metabolismo , Fase S/efeitos dos fármacos , Fase S/genética , Suínos , Fator de Crescimento Transformador beta/farmacologia , Regulação para Cima/fisiologia , Proteínas rap de Ligação ao GTPRESUMO
Stimulation of smooth muscle cells with basic fibroblast growth factor (bFGF) results in the activation of the mitogen-activated protein kinase (MAP kinase) cascade and leads to cell proliferation. We show that transforming growth factor beta 1 (TGF-beta 1), at concentrations that completely inhibited bFGF-induced mitogenic activity, decreased bFGF-induced MAP kinase activity. Under these conditions, tyrosine and threonine phosphorylations of MAP kinase were differentially affected depending on the time period of TGF-beta 1 pretreatment. After a short (30 min) TGF-beta 1 pretreatment, the bFGF-mediated increase in phosphorylation of p42mapk on threonine was inhibited, with no effect on the level of phosphotyrosine or decrease in the electrophoretic mobility of p42mapk. This suggests that TGF-beta 1 inhibited MAP kinase activity through the action of a serine/threonine phosphatase. In contrast, a longer TGF-beta 1 pretreatment (4 h) partly inhibited the bFGF-induced MAP kinase mobility shift and correlated with the inhibition of phosphorylation on both threonine and tyrosine, suggesting that long-term TGF-beta 1 treatment prevented activation of the MAP kinase cascade or directly blocked MAP kinase. The ability of long-term (4 h) but not short-term (30 min) TGF-beta 1 pretreatment to inhibit MAP kinase activity was completely dependent on protein synthesis and suggests that TGF-beta 1 inhibits MAP kinase activity by two distinct mechanisms. These findings provide a molecular basis for the growth-inhibitory action TGF-beta 1 on bFGF-induced mitogenic activity.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/biossíntese , Fator 2 de Crescimento de Fibroblastos/farmacologia , Músculo Liso Vascular/enzimologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Aorta Torácica/enzimologia , Células Cultivadas , Cicloeximida/farmacologia , DNA/biossíntese , Relação Dose-Resposta a Droga , Indução Enzimática , Cinética , Suínos , Fatores de TempoRESUMO
Fibroblast growth factor 1 (FGF1) and 2 (FGF2) bind to two classes of receptors: the high affinity receptors, a family of four known transmembrane tyrosine kinases (FGF R1-R4), and the low affinity receptors, cell surface and basement membrane heparan sulfate proteoglycan (HSPG). During early (first and second) passages of retinal pigmented epithelial (RPE) cells, both FGF1 and FGF2 exhibited low mitogenic activity, while in later (fifth to ninth) passages the activity of FGF1 remained constant but FGF2 activity increased two- to threefold. We have investigated aspects of FGF receptor interactions and the role of heparin/heparan sulfate which modulates FGF activity on RPE cells during in vitro senescence. Northern blot analysis demonstrated that FGF receptor type 1 (FGF R1) is the major high affinity receptor expressed in RPE cells and that its level of expression did not change during serially passage. Both the FGF R1 and the FGF low affinity receptors' binding characteristics (i.e., Kd and number of sites per cell) for FGF1 were unaffected by passage number, whereas the capacity of FGF2 binding to FGF R1 and to the low affinity receptors increased by two- and fivefold, respectively, in late passages, although the affinities were unchanged. This change in the capacity of FGF2 to bind to FGF R1 and to HSPG was not due to a switch of the IIIc splice form of FGF R1 to the IIIb splice form since the exon IIIc was the most predominant splice form of FGF R1 during RPE cell cultures. Furthermore the ratio of the IIIb to the IIIc splice form was not modified during cell subcultures. In parallel in the older RPE cell passages, expression of perlecan, the major FGF low affinity binding site localized on the extracellular matrix of RPE cells, was much elevated compared to early RPE cell passages. Moreover, the cell surface of late passage RPE cells had 79% more HSPG than early passage cells. Therefore, it is suggested that the increase in the number of FGF low affinity receptors present on the cell surface or basement membrane could account for a part of the greater proliferative response of aged RPE cells to FGF2.
Assuntos
Fator 2 de Crescimento de Fibroblastos/farmacologia , Heparitina Sulfato/metabolismo , Mitógenos/farmacologia , Proteoglicanas/metabolismo , Processamento Alternativo/fisiologia , Animais , Sequência de Bases , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Bovinos , Divisão Celular/fisiologia , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Relação Dose-Resposta a Droga , Regulação para Baixo/fisiologia , Matriz Extracelular/metabolismo , Fator 1 de Crescimento de Fibroblastos/metabolismo , Fator 1 de Crescimento de Fibroblastos/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Expressão Gênica/fisiologia , Proteoglicanas de Heparan Sulfato , Heparina/farmacologia , Heparitina Sulfato/genética , Imuno-Histoquímica , Radioisótopos do Iodo , Cinética , Proteínas de Membrana/metabolismo , Mitógenos/metabolismo , Dados de Sequência Molecular , Epitélio Pigmentado Ocular/citologia , Epitélio Pigmentado Ocular/ultraestrutura , Proteoglicanas/genética , RNA Mensageiro/metabolismo , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/ultraestrutura , Sulfatos/metabolismo , Radioisótopos de Enxofre/metabolismoRESUMO
Basic fibroblast growth factor (bFGF) was internalized by smooth muscle cells (SMC) from pig aorta. Correlation between heparin inhibition of binding and late internalization (8 h) implicated low-affinity sites in bFGF internalization. Transforming growth factor-beta 1 (TGF-beta 1) induced a 38% increase in bFGF internalized between 4 and 8 h. While bFGF and/or TGF-beta 1 enhanced cell-surface proteoglycan synthesis, 35S-labelled proteoglycans of the extracellular matrix (ECM) were not affected. This might be explained by the different turnover rates displayed by the two populations of proteoglycans. Although bFGF and/or TGF-beta 1 induced a similar stimulation in cell-surface chondroitin sulphate/dermatan sulphate and heparan sulphate (HS) proteoglycan synthesis, only the turnover of HS proteoglycans was increased. Twice as much HS proteoglycan was internalized in the presence of TGF-beta 1 or bFGF. Furthermore, TGF-beta 1 induced a 43 +/- 12% increase in HS proteoglycan internalized in the presence of bFGF with a parallel 38% increase in bFGF internalization. Overall, the results indicated that bFGF bound to two HS proteoglycan populations. bFGF storage (70% of bFGF bound to SMC) was not affected by TGF-beta 1 under our conditions and involved ECM proteoglycans characterized by a low turnover. bFGF internalization up-regulated by TGF-beta 1 involved cell-surface HS proteoglycan characterized by a high turnover.
Assuntos
Endocitose , Fator 2 de Crescimento de Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Proteínas de Membrana/metabolismo , Músculo Liso Vascular/metabolismo , Proteoglicanas/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Animais , Aorta Torácica , Células Cultivadas , Relação Dose-Resposta a Droga , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Músculo Liso Vascular/citologia , Suínos , Regulação para CimaRESUMO
Pachydermoperiostosis or primary hypertrophic osteoarthropathy (HOA) is a rare congenital growth disorder of connective tissue. We report a case of severe myelofibrosis in a patient with HOA. When cultured in vitro, patient bone marrow-derived fibroblasts displayed a high proliferative potential with a shortened doubling time (24 hours v 36 to 48 hours for normal fibroblasts). The role of platelet-derived growth factor (PDGF), previously implicated in the pathogenesis of secondary acquired myelofibrosis, was studied. HOA fibroblasts expressed an increased number of PDGF-BB binding sites (300,000 sites/cell v 200,000 sites/cell for normal fibroblasts) without any modification of affinity. The increased expression of PDGF-R beta appeared to result from an accelerated rate of PDGF-R beta resynthesis with normal kinetics of endocytosis. As a consequence, a several-fold increase of PDGF-R beta tyrosine kinase activity was observed. No autocrine mechanism of growth was suspected as neither spontaneous PDGF-R beta autophosphorylation nor mitogenic activity in HOA fibroblast-conditioned medium was detected. Patient serum and platelet lysate were less potent than controls in inducing [3H]thymidine incorporation into HOA fibroblasts. This was inconsistent with a paracrine mechanism of growth. In vitro, human serum or PDGF-BB were not more mitogenic for HOA than normal fibroblasts. High levels of cyclin D1, a putative oncogene, were detected in serum-deprived HOA fibroblasts. Cyclin D1 overexpression could be implicated in the accelerated growth of these cells. Our results suggest that the mechanism of fibroblastic proliferation observed in this case of myelofibrosis might differ from those reported in other acquired myeloproliferative syndromes and could be associated with an intrinsic abnormality of HOA fibroblast growth.
Assuntos
Medula Óssea/patologia , Fibroblastos/patologia , Osteoartropatia Hipertrófica Primária/patologia , Mielofibrose Primária/patologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Anemia/etiologia , Plaquetas/metabolismo , Plaquetas/patologia , Divisão Celular , Células Cultivadas , Ciclina D1 , Ciclinas/biossíntese , Ciclinas/genética , Endocitose , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Oncogênicas/biossíntese , Proteínas Oncogênicas/genética , Oncogenes , Osteoartropatia Hipertrófica Primária/complicações , Fosforilação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Mielofibrose Primária/etiologia , Processamento de Proteína Pós-Traducional , Receptores do Fator de Crescimento Derivado de Plaquetas/análise , Regulação para Cima , beta-Tromboglobulina/metabolismoRESUMO
OBJECTIVES: To investigate the role of platelet activation in the development of systemic sclerosis and the role of interferon-gamma (IFN gamma) in the inhibition of mitogenic activity induced by whole blood serum of patients with systemic sclerosis. METHODS: The mitogenic activity of whole blood serum in the absence or presence of different concentrations of IFN gamma (a potent inhibitor of induced collagen synthesis in dermal fibroblasts) and platelet-poor plasma derived serum were tested on human dermal fibroblasts by measuring incorporation of [3H]thymidine. Platelet activation was determined by quantification of plasma beta-thromboglobulin (beta-TG) using a beta-TG radioimmunoassay kit. RESULTS: The mitogenic activity was significantly increased in whole blood serum and in platelet-poor plasma derived serum of the patients compared with controls. In contrast, no significant increase in beta-TG concentration was observed in scleroderma platelet-poor plasma compared with control. Recombinant human IFN gamma had a greater inhibitory effect on the mitogenic activity induced by whole blood serum of patients than on that produced with control sera, at any concentration of IFN gamma tested. CONCLUSIONS: Our results suggest that mitogenic activity observed in the plasma of sclerodermic patients could originate from cells other than platelets and could be involved in the development of fibrosis. The potent inhibitory effect of IFN gamma on this proliferative activity may account for the beneficial effect of this cytokine in the treatment of progressive systemic sclerosis.
Assuntos
Interferon gama/farmacologia , Mitose/fisiologia , Escleroderma Sistêmico/sangue , Adulto , Síndrome CREST/sangue , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Pessoa de Meia-Idade , Mitose/efeitos dos fármacos , Proteínas Recombinantes , Pele/patologia , Timidina/metabolismo , beta-Tromboglobulina/análiseRESUMO
The effect of platelet-derived growth factor (PDGF) on Rap1 expression was investigated in rat vascular smooth muscle cells (SMC). First, evidence for Rap1 proteins was shown by their: (i) detection in membranes using a specific anti-Rap1 antibody, (ii) typical shift in electrophoretic mobility as a consequence of reduction, and (iii) cAMP-induced phosphorylation and immunoprecipitation. Then, the mitogenic activity of 10 ng/ml PDGF AA and BB for 48 h, resulting in a 2- and 5-fold increase in [3H]thymidine incorporation, was correlated with that of total Rap1 protein expression which was found to be 99% +/- 36% and 260% +/- 70%, respectively. Further time-course studies established that this up-regulation of Rap1 proteins was only observed after 48 h of PDGF BB treatment. Lastly, comparative RT-PCR of both rap1a and rap1b mRNAs showed that PDGF BB also up-regulated the rap1a mRNA species, which was 1.5-fold increased in contrast with the rap1b mRNA species. It is concluded that the PDGF BB-induced SMC proliferation is associated with an up-regulation of Rap1a protein.