RESUMO
Matrix metalloproteinase (MMP) activity is generally associated with normal or pathological extracellular processes such as tissue remodelling in growth and development or in tumor metastasis and angiogenesis. Platelets contain at least three MMPs, 1, 2 and 9 that have been reported to stimulate or inhibit agonist-induced platelet aggregation via extracellular signals. The non-selective Zn+2 chelating MMP inhibitor, 1,10-phenanthroline, and the serine protease inhibitor, AEBSF, were found to inhibit all tested agonist-induced platelet aggregation reactions. In vitro analysis demonstrated that 1,10-phenanthroline completely inhibited MMP-1,2,and 9 but had little to no effect on calpain activity while the converse was true with AEBSF. We now demonstrate that MMP-2 functions intracellularly to regulate agonist-induced platelet aggregations via the hydrolytic activation of talin, the presumed final activating factor of glycoprotein (GP)IIb/IIIa integrin (the inside-out signal). Once activated GPIIb/IIIa binds the dimeric fibrinogen molecule required for platelet aggregation. The active intracellular MMP-2 molecule is complexed with JAK 2/STAT 3, as demonstrated by the fact that all three proteins are co-immunoprecipitated with either anti-JAK 2, or anti-STAT 3 antibodies and by immunofluorescence studies. The MMP-2 platelet activation pathway can be synergistically inhibited with the non-selective MMP inhibitor, 1,10-phenanthroline, plus a JAK 2 inhibitor. This activation pathway is distinct from the previously reported calpain-talin activating pathway. The identification of a new central pathway for platelet aggregation presents new potential targets for drug regulation and furthers our understanding of the complexity of platelet activation mechanisms.
Assuntos
Plaquetas/efeitos dos fármacos , Regulação da Expressão Gênica , Metaloproteinase 2 da Matriz/metabolismo , Ativação Plaquetária , Talina/metabolismo , Adulto , Sítios de Ligação , Plaquetas/metabolismo , Calpaína/metabolismo , Quelantes/química , Dimerização , Humanos , Hidrólise , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Fenantrolinas/química , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Fatores de Tempo , Zinco/químicaRESUMO
An estimated 12% of women in the United States suffer from some form of infertility. In vitro fertilization (IVF) is the most common treatment for infertility encompassing over 99% of all assisted reproductive technologies. However, IVF has a low success rate. Live birth rates using IVF can range from 40% in women younger than 35 years to 4% in women older than 42 years. Costs for a successful IVF outcome can be upward of $61,000. The low success rate of IVF has been attributed to the inability of the blastocyst to implant to the uterus. Blastocyst implantation is initiated by L-selectin expressing cells, trophoblasts, binding to L-selectin ligands, primarily sialyl Lewis X (sLeX), on the uterine surface endometrium. Legal and ethical considerations have limited the research on human subjects and tissues, whereas animal models are costly or do not properly mimic human implantation biochemistry. In this work, we describe a cellular model system for quantifying L-selectin adhesion mechanics. L-selectin expression was confirmed in Jeg-3, JAR, and BeWo cell lines, with only Jeg-3 cells exhibiting surface expression. Jeg-3 cells were cultured into three-dimensional spheres, termed "trophospheres," as a mimic to human blastocysts. Detachment assays using a custom-built parallel plate flow chamber show that trophospheres detach from sLeX functionalized slides with 2.75 × 10(-3) dyn of force and 7.5 × 10(-5) dyn-cm of torque. This work marks the first time a three-dimensional cell model has been utilized for quantifying L-selectin binding mechanics related to blastocyst implantation.
Assuntos
Implantação do Embrião , Fertilização , Selectina L/metabolismo , Esferoides Celulares/citologia , Trofoblastos/citologia , Adulto , Fenômenos Biomecânicos , Adesão Celular , Linhagem Celular , Feminino , Humanos , Oligossacarídeos/metabolismo , Antígeno Sialil Lewis X , Esferoides Celulares/metabolismo , Trofoblastos/metabolismoRESUMO
Human interleukin-5 receptor α (IL5Rα) is a glycoprotein that contains four N-glycosylation sites in the extracellular region. Previously, we found that enzymatic deglycosylation of IL5Rα resulted in complete loss of IL5 binding. To localize the functionally important carbohydrate moieties, we employed site-directed mutagenesis at the N-glycosylation sites (Asn(15), Asn(111), Asn(196), and Asn(224)). Because Asn-to-Gln mutagenesis caused a significant loss of structural integrity, we used diverse mutations to identify stability-preserving changes. We also rationally designed mutations at and around the N-glycosylation sites based on sequence alignment with mouse IL5Rα and other cytokine receptors. These approaches were most successful at Asn(15), Asn(111), and Asn(224). In contrast, any replacement at Asn(196) severely reduced stability, with the N196T mutant having a reduced binding affinity for IL5 and diminished biological activity because of the lack of cell surface expression. Lectin inhibition analysis suggested that the carbohydrate at Asn(196) is unlikely involved in direct ligand binding. Taking this into account, we constructed a stable variant, with triple mutational deglycosylation (N15D, I109V/V110T/N111D, and L223R/N224Q). The re-engineered protein retained Asn(196) while the other three glycosylation sites were eliminated. This mostly deglycosylated variant had the same ligand binding affinity and biological activity as fully glycosylated IL5Rα, thus demonstrating a unique role for Asn(196) glycosylation in IL5Rα function. The results suggest that unique carbohydrate groups in multiglycosylated receptors can be utilized asymmetrically for function.
Assuntos
Asparagina/química , Asparagina/genética , Subunidade alfa de Receptor de Interleucina-5/química , Subunidade alfa de Receptor de Interleucina-5/genética , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Animais , Asparagina/fisiologia , Configuração de Carboidratos , Linhagem Celular , Drosophila melanogaster , Variação Genética , Glicosilação , Humanos , Subunidade alfa de Receptor de Interleucina-5/fisiologia , Ligantes , Camundongos , Dados de Sequência Molecular , Ligação Proteica/genéticaRESUMO
Human platelets are differentially activated by varying concentrations of alpha-thrombin or by beta- and gamma-thrombin via three thrombin receptors, PAR-1, PAR-4 and GPIbalpha.It is likely that the development of a normal or abnormal hemostatic event in humans is dictated, in part, by the selective activation of these receptors. The ability to differentially inhibit these thrombin receptors could, therefore, have clinical significance. We have previously demonstrated that histone H1 selectively inhibits the PAR-4 receptor. In the current study we investigated whether five subtypes of the H1 molecule or fragments of the H1.3 subtype differentially inhibited the PAR-4 receptor. PAR-4 inhibition by all H1 subtypes was saturated at 1 uM with no statistical difference observed with the five H1 subtypes tested. Of the five fragments generated from the H1.3 molecule only one had significant inhibitory activity against PAR-4. The C-terminal fragment, N.1, generated by the proteolysis of the parent molecule by Asp-N endoproteinase (Aeromonas proteolytica) at the single aspartate residue, showed the same level of PAR-4 inhibition as the intact H1.3 at 1 uM concentrations. Removal of two N-terminal amino acids (Asp-Val as determined by MALDI analysis) from the N.1 fragment further enhanced its inhibitory activity. These studies may help to develop specific drugs to differentially inhibit the platelet thrombin receptors.
Assuntos
Histonas/farmacologia , Fragmentos de Peptídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Trombina/farmacologia , Células Sanguíneas , Relação Dose-Resposta a Droga , Humanos , Cinética , Receptores de Trombina/antagonistas & inibidoresRESUMO
Bacillus anthracis has recently been shown to secrete a potently hemolytic/cytolytic protein that has been designated anthrolysin O (ALO). In this work, we initiated a study of this potential anthrax virulence factor in an effort to understand the membrane-binding properties of this protein. Recombinant anthrolysin O (rALO35-512) and two N-terminally truncated versions of ALO (rALO390-512 and rALO403-512) from B. anthracis were overproduced in Escherichia coli and purified to homogeneity. The role of cholesterol in the cytolytic activity of ALO was probed in cellular cholesterol depletion assays using mouse and human macrophage-like lines, and also Drosophila Schneider 2 cells. Challenging the macrophage cells with rALO35-512, but not rALO390-512 or rALO403-512, resulted in cell death by lysis, with this cytolysis being abolished by depletion of the membrane cholesterol. Drosophila cells, which contain ergosterol as their major membrane sterol, were resistant to rALO-mediated cytolysis. In order to determine the molecular mechanism of this resistance, the interaction of rALO with model membranes comprised of POPC alone, or with a variety of structurally similar sterols including ergosterol, was probed using Biacore. Both rALO35-512 and rALO403-512 demonstrated robust binding to model membranes composed of POPC and cholesterol, with amount of protein bound proportional to the cholesterol content. Ergosterol supported greatly reduced binding of both rALO35-512 and rALO403-512, whereas other sterols tested did not support binding. The rALO403-512--membrane interaction demonstrated an equilibrium dissociation constant (KD) in the low nanomolar range, whereas rALO35-512 exhibited complex kinetics likely due to the multiple events involved in pore formation. These results establish the pivotal role of cholesterol in the action of rALO. The biosensor method developed to measure ALO recognition of cholesterol in a membrane environment could be extended to provide a platform for the screening of inhibitors of other membrane-binding proteins and peptides.
Assuntos
Bacillus anthracis/química , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Citotoxinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/química , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colesterol/deficiência , Drosophila melanogaster , Proteínas Hemolisinas , Humanos , Cinética , Glicoproteínas de Membrana/farmacologia , Camundongos , Modelos Biológicos , Proteínas Recombinantes/biossíntese , TemperaturaRESUMO
The mechanisms of cell death and the progressive degeneration of neural tissue following traumatic brain injury (TBI) have come under intense investigation. However, the complex interactions among the evolving pathologies in multiple cell types obscure the causal relationships between the initial effects of the mechanical trauma at the cellular level and the long-term dysfunction and neuronal death. We used an in vitro model of neuronal injury to study the mechanisms of cell death in response to a well-defined mechanical insult and found that the majority of dead cells were apoptotic. We have previously reported that promotion of membrane repair acutely with the non-ionic surfactant poloxamer 188 (P188) restored cell viability to control values at 24 h postinjury. Here, we showed that P188 significantly inhibits apoptosis and prevents necrosis. We also examined the role of mitogen-activated protein kinases (MAPKs) in cell death. There was a rapid, transient activation of extracellular signal-regulated kinases, c-Jun N-terminal kinase, and p38s after mechanical insult. Of these, activation of the proapoptotic p38 was the greatest. Treatment with P188 inhibited p38 activation; however, direct inhibition of p38 by SB203580, which selectively inhibits the activity of the p38 MAPK, provided only partial inhibition of apoptosis and had no effect on necrosis. These data suggest that multiple signaling pathways may be involved in the long-term response of neurons to mechanical injury. Furthermore, that the membrane resealing action of P188 provides such significant protection from both necrosis and apoptosis suggests that acute membrane damage due to trauma is a critical precipitating event that is upstream of the many signaling cascades contributing to the subsequent pathology.
Assuntos
Apoptose/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Poloxâmero/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/patologia , Ativação Enzimática , Imidazóis/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Necrose/patologia , Neurônios/citologia , Neurônios/metabolismo , Células PC12 , Piridinas/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
After exposure to subtoxic doses of heavy metals such as mercury, H-2(s) mice develop an autoimmune syndrome consisting of the rapid production of IgG autoantibodies that are highly specific for nucleolar autoantigens and a polyclonal increase in serum IgG1 and IgE. In this study, we explore the role of one of the members of the CD28-B7 costimulation families, ICOS-B7 homologous protein (B7h), in the regulation of mercury-induced autoimmunity. The expression of ICOS on T cells was more enhanced in susceptible A.SW mice than in non-responsive C57BL/6 and DBA/2 mice after HgCl(2) treatment. Furthermore, in A.SW mice treated with HgCl(2), administration of a blocking anti-ICOS Ab effectively inhibited anti-nucleolar autoantibodies and total serum IgE production. Taken together, these results indicate that the ICOS-B7h costimulation pathway is required for this autoimmune syndrome and suggest that targeting this pathway might have therapeutic benefits for human autoimmune diseases.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Doenças Autoimunes/induzido quimicamente , Antígeno B7-1/metabolismo , Glicoproteínas de Membrana/metabolismo , Cloreto de Mercúrio/toxicidade , Animais , Anticorpos Antinucleares/biossíntese , Anticorpos Bloqueadores/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Antígenos CD/fisiologia , Antígenos de Diferenciação de Linfócitos T/biossíntese , Antígenos de Diferenciação de Linfócitos T/imunologia , Doenças Autoimunes/genética , Doenças Autoimunes/prevenção & controle , Antígeno B7-1/fisiologia , Antígeno B7-2 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Regulação para Baixo/imunologia , Feminino , Predisposição Genética para Doença , Tolerância Imunológica/genética , Imunoglobulina E/biossíntese , Imunoglobulina G/biossíntese , Proteína Coestimuladora de Linfócitos T Induzíveis , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBARESUMO
This chapter deals with experimental protocols and considerations related to the culture of epithelial cells under anchorage-independent conditions in liquid media. This technique has proven to be a powerful tool in studying the effects of loss of extracellular matrix interaction on crucial aspects of epithelial cell biology. Specifically, examining cells in the absence of substrate attachment, as described in this chapter, will allow the investigator to study the effect of growth factors independently of cell adhesion. Several methods are discussed relating to the preparation of tissue culture plates for suspension cultures and to the choice of a suitable cell culture medium.
Assuntos
Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Células Epidérmicas , Queratinócitos/citologia , Técnicas de Cultura de Células , Células Cultivadas , Meios de Cultivo Condicionados/química , HumanosRESUMO
The human metastasis-associated gene (MTA1), a member of the nucleosome remodeling complex with histone deacetylase activity, is frequently overexpressed in biologically aggressive epithelial neoplasms. Here, we extend this observation to squamous carcinoma cells, which express high levels of MTA1 relative to normal or immortalized keratinocytes. To address functional aspects of MTA1 expression, we established variants of human immortalized keratinocytes (HaCaT cells) by expressing MTA1 cDNA in both the sense and antisense orientations. We demonstrate that (1) forced MTA1 expression enhances migration and invasion of immortalized keratinocytes; (2) MTA1 expression is necessary but not sufficient for cell survival in the anchorage independent state; (3) MTA1 contributes to expression of the anti-apoptotic Bcl-2 family member Bcl-x(L); (4) MTA1 expression in immortalized keratinocytes depends, in part, on activation of the epidermal growth factor receptor (EGFR). These results establish that, in keratinocytes, MTA1 expression contributes to several aspects of the metastatic phenotype including survival in the anchorage independent state, migration, and invasion.