RESUMO
The assay that combines rapid-cycle PCR with allele-specific fluorescent probe melting profiles performed on the Roche Diagnostics LightCycler is commonly employed for genotyping the HFE gene. We report three illustrative cases of the pros and cons of this method. In two cases, atypical melting curves allows the identification of new DNA substitutions in the HFE gene, whereas, in the third case, a typical melting curve of c.845G>A mutation (C282Y) homozygosity overlooks a nucleotide change and promotes misdiagnosis of HH.
Assuntos
Análise Mutacional de DNA/métodos , Hemocromatose/diagnóstico , Antígenos de Histocompatibilidade Classe I/genética , Proteínas de Membrana/genética , Transição de Fase , Adulto , Análise Mutacional de DNA/instrumentação , Erros de Diagnóstico , Genótipo , Proteína da Hemocromatose , Temperatura Alta , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
We have characterised the Procoagulant activity (PCA) of six well-established cell lines by assays of tissue factor (TF), thrombin and FXa generation, flow cytometry using Annexin V, and by binding studies with Factors VIII and IXa. The monocytic (THP-1 & U937) and promyelocytic (NB4) cells expressed high concentrations of TF antigen and activity, whereas TF in the lymphocytic cells (Molt 4, Jurkat & Nalm 6) was very low or absent. However the T-lymphoblastoid cells (Molt 4 & Jurkat) promoted the generation of large amounts of thrombin despite their low TF content, and these cells were also the most active in supporting Factor Xa generation. Molt 4 cells bound Factors VIII and IXa with high capacity and their activity was inhibited by Annexin V. These results indicate that the PCA of T-lymphoblastoid lines is due to expression of negatively charged phospholipids. Flow cytometry studies showed Annexin V binding to the major population of nonapoptotic Molt 4 cells and the PCA of Molt 4 was not increased when apoptosis was induced by staurosporine, indicating that PCA is independent of apoptotic status.
Assuntos
Fosfolipídeos/metabolismo , Linfócitos T/ultraestrutura , Trombofilia/etiologia , Ânions/metabolismo , Apoptose , Membrana Celular/metabolismo , Fator IXa/metabolismo , Fator VIII/metabolismo , Humanos , Fosfatidilserinas/metabolismo , Ligação Proteica , Linfócitos T/metabolismo , Linfócitos T/patologia , Trombofilia/patologia , Tromboplastina/metabolismo , Células Tumorais CultivadasRESUMO
Acute promyelocytic leukaemia (APL) may be associated with disseminated intravascular coagulation, as a result of increased tissue factor (TF) expression and reduced thrombomodulin (TM) expression by APL blast cells. During retinoid acid (RA)- and dibutyryl cAMP (dbcAMP)-induced differentiation of the APL cells, there is a marked up-modulation of both the protein kinase A (PKA) and C (PKC) activities. In order to further assess whether these kinases are intimately associated with both the differentiation process and the regulation of TF and TM expression, we have correlated the modulation of their respective pathways with the extent of differentiation and modulation of these cellular receptors. NB4 cells were incubated with all-trans-RA (ATRA) or dbcAMP for up to 48 h. The contribution of phospholipase C (PLC), inositol phosphate (IP), PKC and PKA in the expression of CD11b, TF and TM was studied by the use of specific inhibitors. Myo-inositol uptake and PKC activity increased in cells induced to differentiate by ATRA but the retinoid did not affect cAMP levels or PKA activity. Under treatment with dbcAMP, PKA activity was increased while inositol uptake and PKC activity remained unchanged. Our results show that the effects of ATRA and dbcAMP on promyelocytic cells are closely related, respectively, to the PLC/IP/PKC and the cAMP/PKA pathways. In cells induced to differentiate by ATRA, CD11b expression seems more closely related to inositol uptake than to PKC activity while the expression of TF and TM show the opposite pattern, which suggests cellular events regulated at a different level within a common signal transduction pathway.
Assuntos
Granulócitos/efeitos dos fármacos , Transdução de Sinais , Trombomodulina/metabolismo , Tromboplastina/metabolismo , Bucladesina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Granulócitos/citologia , Granulócitos/metabolismo , Humanos , Leucemia Promielocítica Aguda/metabolismo , Antígeno de Macrófago 1/efeitos dos fármacos , Antígeno de Macrófago 1/metabolismo , Proteína Quinase C/metabolismo , Proteína Quinase C/farmacocinética , Proteína Quinase C/fisiologia , Trombomodulina/efeitos dos fármacos , Tromboplastina/efeitos dos fármacos , Tretinoína/farmacologia , Células Tumorais CultivadasRESUMO
When Glu-plasminogen is bound to cells, plasmin (Pm) formation by plasminogen (Pg) activators is markedly enhanced compared with the reaction in solution. It is not known whether the direct activation of Glu-Pg by Pg activators is promoted on the cell surface or whether plasminolytic conversion of Glu-Pg to the more readily activated Lys-Pg is necessary for enhanced Pm formation on the cell surface. To distinguish between these potential mechanisms, we tested whether Pm formation on the cell surface could be stimulated in the absence of conversion of Glu-Pg to Lys-Pg. Rates of activation of Glu-Pg, Lys-Pg, and a mutant Glu-Pg, [D646E]Glu-Pg, by either tissue Pg activator (t-PA) or urokinase (u-PA) were compared when these Pg forms were either bound to human umbilical vein endothelial cells (HUVEC) or in solution. ([D646E]Glu-Pg can be cleaved at the Arg(561)-Val(562) bond by Pg activators but does not possess Pm activity subsequent to this cleavage because of the mutation of Asp(646) of the serine protease catalytic triad.) Glu-Pg activation by t-PA was enhanced on HUVEC compared with the solution phase by 13-fold. In contrast, much less enhancement of Pg activation was observed with [D646E]Glu-Pg ( approximately 2-fold). Although the extent of activation of Lys-Pg on cells was similar to that of Glu-Pg, the cells afforded minimal enhancement of Lys-Pg activation compared with the solution phase (1.3-fold). Similar results were obtained when u-PA was used as activator. When Glu-Pg was bound to the cell in the presence of either t-PA or u-PA, conversion to Lys-Pg was observed, but conversion of ([D646E]Glu-Pg to ([D646E]Lys-Pg was not detected, consistent with the conversion of Glu-Pg to Lys-Pg being necessary for optimal enhancement of Pg activation on cell surfaces. Furthermore, we found that conversion of [D646E]Glu-Pg to [D646E]Lys-Pg by exogenous Pm was markedly enhanced ( approximately 20-fold) on the HUVEC surface, suggesting that the stimulation of the conversion of Glu-Pg to Lys-Pg is a key mechanism by which cells enhance Pg activation.
Assuntos
Membrana Celular/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Fibrinolíticos/metabolismo , Fragmentos de Peptídeos/metabolismo , Plasminogênio/metabolismo , Movimento Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Humanos , Mutação , Fragmentos de Peptídeos/fisiologia , Plasminogênio/fisiologia , Ativadores de Plasminogênio/metabolismo , Ligação Proteica , Serina Endopeptidases/metabolismo , Fatores de Tempo , Ativador de Plasminogênio Tecidual/metabolismo , Cordão Umbilical/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
To test the hypothesis that Glu202, adjacent to the His201 residue that participates in the coordination of Zn(2+) in matrix metalloproteinase-3 (MMP-3 or stromelysin-1), plays a role in its enzymatic activity it was substituted with Ala, Lys or Asp by site-specific mutagenesis. Wild-type proMMP-3, proMMP-3(E202A), proMMP-3(E202K) and proMMP-3(E202D) were expressed in Escherichia coli and purified to apparent homogeneity. Whereas 33-kDa wild-type proMMP-3 (consisting of the propeptide and catalytic domains) was quantitatively converted to 24-kDa active MMP-3 by treatment with p-aminophenyl-mercuric acetate (APMA), proMMP-3(E202A) and proMMP-3 (E202K) were fully resistant to APMA and proMMP-3 (E202D) was quantitatively converted into a 14-kDa species. In contrast, treatment with plasmin quantitatively converted the wild-type and the three mutant proMMP-3 moieties into the corresponding 24-kDa MMP-3 moieties. Biospecific interaction analysis revealed comparable affinity for binding to plasminogen of wild-type and mutant proMMP-3 (K(a) of 2.6-6.3 x 10(6) M(-1)) or MMP-3 (K(a) of 33-58 x 10(6) M(-1)) moieties. The affinity for binding to single-chain urokinase-type plasminogen activator (scu-PA) was also similar for wild-type and mutant proMMP-3 (K(a) of 5.0-6.9 x 10(6) M(-1)) or MMP-3 (K(a) of 37-72 x 10(6) M(-1)) moieties. However, MMP-3(E202A) and MMP-3(E202K) did not hydrolyze plasminogen whereas MMP-3(E202D) showed an activity of 20--30% of wild-type MMP-3. All three mutants were inactive towards scu-PA under conditions where this was quantitatively cleaved by wild-type MMP-3. Furthermore, MMP-3(E202A) and MMP-3(E202K) were inactive toward a fluorogenic substrate and MMP-3 (E202D) displayed about 15% of the activity of wild-type MMP-3. Taken together, these data suggest that Glu202 plays a crucial role in the enzymatic activity of MMP-3.
Assuntos
Ácido Glutâmico/fisiologia , Metaloproteinase 3 da Matriz/metabolismo , Acetato de Fenilmercúrio/análogos & derivados , Alanina/química , Ácido Aspártico/química , Sítios de Ligação , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Escherichia coli/metabolismo , Fibrinolisina/farmacologia , Ácido Glutâmico/química , Humanos , Hidrólise , Cinética , Lisina/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Acetato de Fenilmercúrio/farmacologia , Plasminogênio/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Fatores de Tempo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Zinco/metabolismoRESUMO
Plasminogen plays an integral role in the inflammatory response, and this participation is likely to depend on its interaction with cell surfaces. It has previously been reported that isolation of human neutrophils from blood leads to a spontaneous increase in their plasminogen-binding capacity, and the basis for this up-regulation has been explored as a model for mechanisms for modulation of plasminogen receptor expression. Freshly isolated human peripheral blood neutrophils exhibited relatively low plasminogen binding, but when cultured for 20 hours, they increased this capacity dramatically, up to 50-fold. This increase was abolished by soybean trypsin inhibitor and was susceptible to carboxypeptidase B treatment, implicating proteolysis and exposure of carboxy-terminal lysines in the enhanced interaction. In support of this hypothesis, treatment of neutrophils with elastase, cathepsin G, or plasmin increased their plasminogen binding, and specific inhibitors of elastase and cathepsin G suppressed the up-regulation that occurred during neutrophil culture. When neutrophils were stimulated with phorbol ester, their plasminogen binding increased rapidly, but this increase was insensitive to the protease inhibitors. These results indicate that plasminogen binding to neutrophils can be up-regulated by 2 distinct pathways. A major pathway with the propensity to markedly up-regulate plasminogen binding depends upon the proteolytic remodeling of the cell surface. In response to thioglycollate, neutrophils recruited into the peritoneum of mice were shown to bind more plasminogen than those in peripheral blood, suggesting that modulation of plasminogen binding by these or other pathways may also occur in vivo.
Assuntos
Neutrófilos/metabolismo , Plasminogênio/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Ácido Aminocaproico/farmacologia , Animais , Aprotinina/farmacologia , Carboxipeptidase B , Carboxipeptidases/farmacologia , Catepsina G , Catepsinas/antagonistas & inibidores , Catepsinas/farmacologia , Linhagem Celular , Células Cultivadas/efeitos dos fármacos , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Fibrinolisina/farmacologia , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/farmacologia , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/efeitos dos fármacos , Peritonite/induzido quimicamente , Peritonite/patologia , Inibidores de Proteases/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteínas/farmacologia , Explosão Respiratória , Serina Endopeptidases , Serpinas , Relação Estrutura-Atividade , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Ativador de Plasminogênio Tecidual/farmacologia , Inibidor da Tripsina de Soja de Kunitz/farmacologia , Células U937RESUMO
Matrix metalloproteinase-3 (MMP-3 or stromelysin-1) specifically binds to tissue-type plasminogen activator (t-PA), without however, hydrolyzing the protein. Binding affinity to proMMP-3 is similar to single chain t-PA, two chain t-PA and active site mutagenized t-PA (Ka of 6.3 x 106 to 8.0 x 106 M-1), but is reduced for t-PA lacking the finger and growth factor domains (Ka of 2.0 x 106 M-1). Activation of native Glu-plasminogen by t-PA in the presence of proMMP-3 obeys Michaelis-Menten kinetics; at saturating concentrations of proMMP-3, the catalytic efficiency of two chain t-PA is enhanced 20-fold (kcat/Km of 7.9 x 10-3 vs. 4.1 x 10-4 microM-1.s-1). This is mainly the result of an enhanced affinity of t-PA for its substrate (Km of 1.6 microM vs. 89 microM in the absence of proMMP-3), whereas the kcat is less affected (kcat of 1.3 x 10-2 vs. 3.6 x 10-2 s-1). Activation of Lys-plasminogen by two chain t-PA is stimulated about 13-fold at a saturating concentration of proMMP-3, whereas that of miniplasminogen is virtually unaffected (1.4-fold). Plasminogen activation by single chain t-PA is stimulated about ninefold by proMMP-3, whereas that by the mutant lacking finger and growth factor domains is stimulated only threefold. Biospecific interaction analysis revealed binding of Lys-plasminogen to proMMP-3 with 18-fold higher affinity (Ka of 22 x 106 M-1) and of miniplasminogen with fivefold lower affinity (Ka of 0.26 x 106 M-1) as compared to Glu-plasminogen (Ka of 1.2 x 106 M-1). Plasminogen and t-PA appear to bind to different sites on proMMP-3. These data are compatible with a model in which both plasminogen and t-PA bind to proMMP-3, resulting in a cyclic ternary complex in which t-PA has an enhanced affinity for plasminogen, which may be in a Lys-plasminogen-like conformation. Maximal binding and stimulation require the N-terminal finger and growth factor domains of t-PA and the N-terminal kringle domains of plasminogen.
Assuntos
Precursores Enzimáticos/metabolismo , Metaloendopeptidases/metabolismo , Plasminogênio/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Ativação Enzimática , Precursores Enzimáticos/isolamento & purificação , Fibrinolisina/antagonistas & inibidores , Fibrinolisina/metabolismo , Humanos , Cinética , Kringles , Metaloendopeptidases/isolamento & purificação , Plasminogênio/química , Ligação Proteica , Especificidade por Substrato , Ativador de Plasminogênio Tecidual/químicaRESUMO
The chemokine macrophage inflammatory protein (MIP)-2alpha was identified as a plasminogen binding protein by phage display analysis. MIP-2alpha and a truncated form lacking 5 lysine residues in the COOH-terminal region (mut-MIP-2alpha) were expressed in E. coli and purified to apparent homogeneity. Purified MIP-2alpha but not mut-MIP-2alpha bound specifically to plasminogen, with K(A) of 3.7 X 10(5) M(-1) for the interaction of plasminogen with surface-bound MIP-2alpha. Binding and competition experiments indicated that the interaction involves the region comprising the first 3 kringles of plasminogen and the COOH-terminal lysine-rich domain of MIP-2alpha. Activation of plasminogen bound to surface-associated MIP-2alpha by two-chain urokinase-type plasminogen activator (tcu-PA) was about 2.5-fold more efficient than in solution (catalytic efficiency k(cat)K(M) of 0.1 microM(-1)s(-1), as compared to 0.04 microM(-1)s(-1). In contrast, binding of plasminogen to MIP-2alpha in solution was very weak, as evidenced by the absence of competition of MIP-2alpha with lysine-Sepharose or with human THP-1 cells for binding of plasminogen. In agreement with this finding, addition of excess MIP-2alpha did not affect the main functional properties of plasmin(ogen) in solution, as indicated by unaltered activation rates of plasminogen by tcu-PA or tissue-type plasminogen activator (t-PA), t-PA-mediated fibrinolysis, and inhibition rate of plasmin by alpha2-antiplasmin. Thus, association of MIP-2alpha with surfaces exposes its COOH-terminal plasminogen-binding site, and may result in enhanced local plasmin generation.
Assuntos
Monocinas/metabolismo , Plasminogênio/metabolismo , Sítios de Ligação , Quimiocina CXCL2 , Quimiocinas CXC , Endotélio Vascular/citologia , Ativação Enzimática/efeitos dos fármacos , Fibrinólise/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , Kringles , Leucemia Monocítica Aguda/patologia , Lisina/metabolismo , Macrófagos/metabolismo , Monocinas/química , Monocinas/genética , Monocinas/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Ativador de Plasminogênio Tecidual/farmacologia , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/farmacologiaRESUMO
The 55-kD urokinase (uPA) receptor (uPAR, CD87) is capable of binding uPA and may be involved in regulating cell-associated plasminogen activation and pericellular proteolysis. While investigating the relationship between uPAR levels and plasmin generation, we found that uPA-catalyzed plasminogen activation is stimulated by cells which do not express uPAR. This uPAR-independent mechanism appears to be at least as effective in vitro as uPAR-dependent stimulation, such that stimulation on the order of 30-fold was observed, resulting from improvements in both apparent kcat and apparent Km. The mechanism depends on simultaneous binding of both uPA and plasminogen to the cell and requires the presence of the amino-terminal fragment (ATF), available in single chain and two chain high-molecular-weight uPA, but not low-molecular-weight uPA. Stimulation was observed in all leukemic cell lines investigated at similar optimum concentrations of 10(6) to 10(7) cells/mL and may be more general. A mechanism is proposed whereby uPA can associate with binding sites on the cell surface of lower affinity, but higher capacity than uPAR, but these are sufficient to stimulate plasmin generation even at subphysiologic uPA concentrations. This mechanism is likely to operate under conditions commonly used for in vitro studies and may have some significance in vivo.
Assuntos
Ativadores de Plasminogênio/metabolismo , Plasminogênio/metabolismo , Receptores de Superfície Celular/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Humanos , Ativadores de Plasminogênio/farmacologia , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Transdução de Sinais , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/farmacologiaRESUMO
A number of cell types have previously been shown to bind tissue plasminogen activator (tPA), which in some cases can remain active on the cell surface resulting in enhanced plasminogen activation kinetics. We have investigated several cultured cell lines, U937, THP1, K562, Molt4, and Nalm6 and shown that they bind both tPA and plasminogen and are able to act as promoters of plasminogen activation in kinetic assays. To understand what structural features of tPA are involved in cell surface interactions, we performed kinetic assays with a range of tPA domain deletion mutants consisting of full-length glycosylated and nonglycosylated tPA (F-G-K1-K2-P), DeltaFtPA (G-K1-K2-P), K2-P tPA (BM 06.022 or Reteplase), and protease domain (P). Deletion variants were made in Escherichia coli and were nonglycosylated. Plasminogen activation rates were compared with and without cells, over a range of cell densities at physiological tPA concentrations, and produced maximum levels of stimulation up to 80-fold with full-length, glycosylated tPA. Stimulation for nonglycosylated full-length tPA dropped to 45-60% of this value. Loss of N-terminal domains as in DeltaFtPA and K2P resulted in a further loss of stimulation to 15-30% of the full-length glycosylated value. The protease domain alone was stimulated at very low levels of up to 2-fold. Thus, a number of different sites are involved in cell interactions especially within finger and kringle domains, which is similar to the regulation of tPA activity by fibrin. A model was developed to explain the mechanism of stimulation and compared with actual data collected with varying cell, plasminogen, or tPA concentrations and different tPA variants. Experimental data and model predictions were generally in good agreement and suggest that stimulation is well explained by the concentration of reactants by cells.
Assuntos
Ativador de Plasminogênio Tecidual/metabolismo , Sequência de Bases , Linhagem Celular , Primers do DNA , Glicosilação , Humanos , Modelos Químicos , Ligação Proteica , Deleção de SequênciaRESUMO
UV irradiation leads to severe damage, such as cutaneous inflammation, immunosuppression, and cancer, but it also results in a gene induction protective response termed the UV response. The signal triggering the UV response was thought to originate from DNA damage; recent findings, however, have shown that it is initiated at or near the cell membrane and transmitted via cytoplasmic kinase cascades to induce gene transcription. Urokinase-type plasminogen activator (uPA) was the first protein shown to be UV inducible in xeroderma pigmentosum DNA repair-deficient human cells. However, the underlying molecular mechanisms responsible for the induction were not elucidated. We have found that the endogenous murine uPA gene product is transcriptionally upregulated by UV in NIH 3T3 fibroblast and F9 teratocarcinoma cells. This induction required an activator protein 1 (AP1) enhancer element located at -2.4 kb, since deletion of this site abrogated the induction. We analyzed the contribution of the three different types of UV-inducible mitogen-activated protein (MAP) kinases (ERK, JNK/SAPK, and p38) to the activation of the murine uPA promoter by UV. MEKK1, a specific JNK activator, induced transcription from the uPA promoter in the absence of UV treatment, whereas coexpression of catalytically inactive MEKK1(K432M) and of cytoplasmic JNK inhibitor JIP-1 inhibited UV-induced uPA transcriptional activity. In contrast, neither dominant negative MKK6 (or SB203580) nor PD98059, which specifically inhibit p38 and ERK MAP kinase pathways, respectively, could abrogate the UV-induced effect. Moreover, our results indicated that wild-type N-terminal c-Jun, but not mutated c-Jun (Ala-63/73), was able to mediate UV-induced uPA transcriptional activity. Taken together, we show for the first time that kinases of the JNK family can activate the uPA promoter. This activation links external UV stimulation and AP1-dependent uPA transcription, providing a transcription-coupled signal transduction pathway for the induction of the murine uPA gene by UV.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica/efeitos da radiação , Proteínas de Membrana/genética , Proteínas Quinases Ativadas por Mitógeno , Transdução de Sinais , Ativador de Plasminogênio Tipo Uroquinase/genética , Células 3T3 , Complexo 1 de Proteínas Adaptadoras , Subunidades alfa do Complexo de Proteínas Adaptadoras , Proteínas Adaptadoras de Transporte Vesicular , Animais , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Cinética , Camundongos , RNA Mensageiro , Transcrição Gênica , Ativação Transcricional , Raios UltravioletaRESUMO
The broad spectrum protease urokinase-type plasminogen activator (uPA) has been implicated in muscle regeneration in vivo as well as in myogenic proliferation and differentiation in vitro. These processes are known to be modulated by basic fibroblast growth factor (FGF-2) and serum. We therefore investigated the mechanism(s) underlying the regulation of uPA expression by these two stimuli in proliferating and differentiating myoblasts. The expression of uPA mRNA and the activity of the uPA gene product were induced by FGF-2 and serum in proliferating myoblasts. uPA induction occurred at the level of transcription and required the uPA-PEA3/AP1 enhancer element, since deletion of this site in the full promoter abrogated induction by FGF-2 and serum. Using L6E9 skeletal myoblasts, devoid of endogenous FGF receptors, which have been engineered to express either FGF receptor-1 (FGFR1) or FGF receptor-4 (FGFR4), we have demonstrated that both receptors, known to be expressed in skeletal muscle cell precursors, were able to mediate uPA induction by FGF-2, whereas serum stimulation was FGF receptor-independent. The induction of uPA by FGF-2 and serum in FGFR1- and in FGFR4-expressing myoblasts required the mitogen-activated protein kinase pathway, since treatment of cells with a specific inhibitor of the mitogen-activated protein kinase/extracellular signal-regulated kinase-2 kinase, PD98059, blocked uPA promoter induction. Although FGF-2 and serum induced uPA in proliferating myoblasts, their actions on cell-cell contact-induced differentiating myoblasts differed dramatically. FGF-2, but not serum, repressed uPA expression in differentiation-committed myoblasts, and these effects were also shown to occur at the level of uPA transcription. Altogether, these results indicate a dual regulation of the uPA gene by FGF-2 and serum, which ensures uPA expression throughout the whole myogenic process in different myoblastic lineages. The effects of FGF-2 and serum on uPA expression may contribute to the proteolytic activity required during myoblast migration and fusion, as well as in muscle regeneration.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Desenvolvimento Muscular , Receptores Proteína Tirosina Quinases , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Animais , Comunicação Celular , Diferenciação Celular , Divisão Celular , Linhagem Celular , Camundongos , Modelos Biológicos , Regiões Promotoras Genéticas , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Fator de Transcrição AP-1/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ativador de Plasminogênio Tipo Uroquinase/genéticaRESUMO
Alpha-enolase is an ubiquitous cytoplasmic glycolytic enzyme which also exhibits cell surface mediated functions and a structural role in the lens of some species. An alpha-enolase related molecule (alpha-ERM) is present on the surfaces of neutrophils, monocytes and monocytoid cells and has the capacity to specifically bind plasminogen, suggesting that alpha-ERM may function as a plasminogen receptor. We have generated a monoclonal antibody (mAB), 9C12, against alpha-ERM. This mAB reacted with both alpha-ERM and purified human alpha-enolase in Western blotting and in enzyme linked immunosorbent assays (ELISA). mAB 9C12 detected a cell surface associated molecule on human peripheral blood neutrophils and on U937 human monocytoid cells as assessed by fluorescence activated cell sorting (FACS) analyses. In addition, mAB 9C12 recognized an intracellular pool of alpha-enolase/alpha-ERM in permeabilized U937 cells. A phage display approach was employed to identify the alpha-enolase epitope recognized by mAB 9C12. Random fragments of 100-300 base pairs (bp), obtained from the full length human alpha-enolase cDNA, were cloned into the filamentous phage vector pComb3B, to generate a phage-displayed peptide library. Recombinant phages binding to mAB 9C12 were selected and their DNA inserts characterized by direct sequencing. All of the fragments which bound to mAB 9C12 encoded the common sequence DLDFKSPDDPSRYISP, spanning amino acids 257-272 of human alpha-enolase. This sequence is located within an external loop of the molecule. These data indicate that this sequence contains the epitope recognized by mAB 9C12 and is, therefore, exposed on the cell surface, further suggesting that alpha-enolase and alpha-ERM share common amino acid sequences.
Assuntos
Epitopos/análise , Fosfopiruvato Hidratase/imunologia , Receptores de Superfície Celular/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais , Sítios de Ligação , Western Blotting , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Neutrófilos/química , Biblioteca de Peptídeos , Fosfopiruvato Hidratase/análise , Conformação Proteica , Receptores de Superfície Celular/análise , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Propriedades de SuperfícieRESUMO
The endothelial cell line ECV304 is a spontaneously transformed cell line established from human umbilical vein. The characterization of tissue factor (TF) expression by ECV304 cells has been accomplished in this study. ECV304 cells expressed both TF mRNA and antigen (TFag) constitutively. In ECV304 cell lysates, the levels of TFag (1.4+/-0.3 ng of TFag/10[6] cells) were considerably higher than in THP-1 monocytoid cells (0.07+/-0.03 ng of TFag/10[6] cells). TFag was also detected on the ECV304 cell surface by flow cytometric studies. In binding analyses, 3.5+/-0.7 x 10(4) molecules of TF per cell were estimated, similar to the amounts found in ECV304 cell lysates (2.9+/-0.6 x 10(4) molecules/cell), suggesting that all TFag was translocated to the cell surface. Phorbol myristate acetate (PMA) stimulation of ECV304 cells resulted in an increase of TF mRNA levels, which was abrogated when gene transcription was impaired, suggesting a transcriptional regulation of the TF gene by PMA. In contrast, TFag was not elevated by PMA-stimulation, indicating the existence of additional posttranscriptional mechanisms. Thus, ECV304 cells constitute a singular endothelial cell model for exploring the regulation of TF expression.
Assuntos
Endotélio Vascular/metabolismo , Tromboplastina/metabolismo , Anticorpos Monoclonais , Linhagem Celular , Membrana Celular/metabolismo , Ácido Edético/farmacologia , Endotélio Vascular/citologia , Humanos , Monócitos/metabolismo , RNA Mensageiro/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Tromboplastina/genética , Fatores de TempoRESUMO
Urokinase-type plasminogen activator (uPA) is one of the components of blood's fibrinolytic cascade. uPA acts as a broad spectrum proteolytic enzyme involved in different physio-pathological processes including cellular fibrinolysis, adhesion, migration, invasion and remodeling. Here, we present evidence that uPA participates in myogenesis, a process which requires drastic cell membrane reorganization, leading to the plurinucleated myotube from the progenitor myoblast. We have dissected the expression of uPA throughout the different myogenic compartments and found an increase in uPA enzymatic activity associated with myotube formation in C2C12 myoblast cells, with uPA mRNA increasing prior the onset of fusion and differentiation. When both fusion and differentiation were blocked by specific inhibitors (DMSO, cytochalasin B) the levels of uPA were strongly downregulated. This process was reversible and specific: the removal of the inhibitors immediately restored the levels of uPA mRNA while the specific inhibition of uPA enzymatic activity by an anti-uPA antibody resulted in a 50% reduction of the extent of fusion and in the abrogation of muscle-specific gene products, such as alpha-actin and MyoD. Moreover, the conversion of fibroblasts to muscle-like cells upon acquisition of MyoD resulted in a dramatic increase of uPA mRNA, which was partially due to transcriptional activation of the uPA gene. These results indicate that the increase in uPA expression prior to fusion and differentiation occurs via a MyoD-mediated mechanism whereas the normal MyoD expression requires the plasminogen activation-dependent activity of this protease. Therefore, these studies extend the sphere of influence of myogenic factors to fibrinolysis, an intrinsic component of the hematological system. Taken together, one mechanism used by the myoblast cell to become a differentiated myotube, involving the inductive extracellular proteolysis of urokinase, is proposed.
Assuntos
Músculos/citologia , Ativador de Plasminogênio Tipo Uroquinase/antagonistas & inibidores , Actinas/metabolismo , Animais , Northern Blotting , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Fusão Celular/efeitos dos fármacos , Linhagem Celular , Citocalasina B/metabolismo , Dimetil Sulfóxido/farmacologia , Fibroblastos/citologia , Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Camundongos , Músculos/fisiologia , Proteína MyoD/fisiologia , Ativadores de Plasminogênio , RNA Mensageiro/metabolismo , Transfecção , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/fisiologiaRESUMO
Tissue factor (TF) and urokinase receptor (uPAR) are key cellular receptors triggering, respectively, coagulation and fibrinolysis. Bleeding complications among leukemic patients have been related to an abnormal expression of TF by blast cells and/or to an abnormal fibrinolytic response. In this study the expression of TF and uPAR has been assessed in 18 acute non-lymphoblastic and 8 lymphoblastic leukemic blast cells using several methodological approaches. TF mRNA was evaluated by in situ hybridization and TF and uPAR antigen were evaluated immunologically in cell lysates and on the cell surface by flow cytometry. In addition, TF-procoagulant activity was measured in coagulation-based assays. The reliability of these methods was corroborated in six leukemic cell lines of different lineages and states of maturation. Disseminated intravascular coagulation was detected in two M3 leukemia patients whose blast cells expressed high amounts of TF. Hyperfibrinolysis was detected in one M1 and two M2 patients, whose blast cells displayed a high content of uPAR antigen, but no TF. Furthermore, M5 leukemia blast cells expressed both TF and uPAR, although no hemostatic defects or bleeding complications were detected in these patients. Taken together, although a limited number of patients was included in this study, these data suggest that in leukemia patients exhibiting bleeding, either TF or uPAR are expressed by their blast cells. However, the presence of these receptors does not necessarily imply the existence of a hemostatic disorder.
Assuntos
Hemorragia/etiologia , Leucemia/sangue , Receptores de Superfície Celular/metabolismo , Tromboplastina/metabolismo , Doença Aguda , Coagulação Sanguínea , Humanos , Leucemia/complicações , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Células Tumorais CultivadasRESUMO
The urinary type plasminogen activator, urokinase (uPA) is localized on the cell surface through the binding of a specific receptor, the uPA receptor (uPAR). The uPA localization enhances plasmin formation on the cell surface and facilitates cell migration. The cellular and tissue distribution of uPAR is not fully established. We have analyzed uPAR expression in nine leukemic cell lines of distinct lineages and maturational states and correlated this with expression of plasminogen receptors, tissue-type plasminogen activator (tPA) receptors and LDL receptor-related protein (LRP). The most immature and least differentiated cell line (an erythro-myeloid cell line) and cells of lymphoid lineage, did not express uPAR, whereas cells differentiated along the myelo-monocytic pathway displayed this receptor. Plasminogen and tPA receptors were expressed by all leukemic cell lines and by all nucleated peripheral blood cells but B and T lymphocytes were negative for cell surface expression of both uPAR and LRP while monocytes and neutrophils were positive for expression of both uPAR and LRP. PMA stimulation induced surface expression of uPAR in lymphocytes but did not induce expression of LRP by these cells. In contrast, lymphoid cell lines were negative for uPAR expression even after PMA stimulation, indicating differences in regulation of uPAR expression between lymphocytes and lymphoid cell lines. The pattern of uPAR expression on leukemic cell lines was also studied on bone marrow blast cells from leukemic patients. Only the most mature myeloid cells expressed uPAR on their surfaces. In contrast, M3 leukemic cells and other blast cells displaying lymphoid markers such as TdT (+) and/or CD2 (+) did not express intracellular or cell-surface associated uPAR, indicating an heterogeneity among these promyelocytic cells and suggesting that uPAR may be a useful marker for leukemia typing. Myeloid blast cells from some patients contained intracellular pools of uPAR but displayed no receptor on the cell surface, suggesting that translocation may be a mechanism regulating uPAR expression in these cells. The comparison of uPAR expression between these cell lines and peripheral blood cells and it correlation with plasminogen receptors, tPA receptors and LRP expression offers new insights regarding potential mechanisms for regulation of uPA-uPAR-mediated pericellular proteolysis.
Assuntos
Leucemia/metabolismo , Leucócitos Mononucleares/metabolismo , Receptores de Superfície Celular/biossíntese , Diferenciação Celular , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia/patologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Receptores Imunológicos/biossíntese , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Ativador de Plasminogênio Tecidual , Células Tumorais CultivadasRESUMO
Plasminogen and tPA bind to a common set of binding sites on nucleated cells. To assess the functional consequences of cellular binding, we have measured the kinetic changes induced by plasminogen activation by tPA on cell surfaces. These studies were carried out with U937 and THP-1 monocytoid cells, with Raji, Nalm6 and Molt4 lymphoid cells and with peripheral blood monocytes and neutrophils. The interactions of plasminogen and tPA with cells induced an increase in the rate of plasmin generation which depended upon the cell concentration. With saturating amounts of U937 monocytoid cells (1.25 x 10(5)/ml) the rate of plasmin generation was 0.39 nM.s-1 versus 0.07 and 0.09 nM.s-1 without cells or without tPA, respectively. The catalytic efficiency of Glu- or Lys-plasminogen activation by tPA increased by 7.2- and 24.2-fold, respectively. These changes were induced by a 72-242-fold reduction in the Km of these interactions which was in the range of 0.3-0.9 microM. These values are below the plasminogen concentration in plasma (1-2 microM). Moreover, we provide new data indicating that 1) only a specific subset of plasminogen binding sites, i.e. molecules exposing carboxyl terminal lysines on the cell surface, promotes plasminogen activation on cells; 2) the first four kringles of plasminogen and the finger of tPA are critical for enhanced plasmin generation on cell surfaces; 3) the simultaneous co-localization of tPA with plasminogen on cell surfaces is required for enhanced plasminogen activation; 4) modulation of plasminogen/tPA receptor expression induces concomitant modulation of the stimulatory effects of cells on plasminogen activation and 5) in a direct comparison, the mechanism by which cells and fibrin fragments accelerate plasminogen activation are similar but not identical. These data suggest that modulation of plasminogen/tPA binding sites permits local and efficient generation of plasmin on cell surfaces.