RESUMEN
Unpublished data from our labs led us to hypothesize that activated protein C (aPC) may initiate an anti-inflammatory signal in endothelial cells by modulating both the integrin αVß3 and protease-activated receptor 2 (PAR2), which may exist in close proximity on the cellular surface. To test this hypothesis and to probe the possible inflammation-related pathway, we designed and synthesized dual-targeting ligands composed of modified versions of two αVß3 ligands and two agonists of PAR2. These novel ligands were connected via copper-catalyzed alkyne-azide cycloadditions with polyethylene glycol (PEG) spacers of variable length. Initial in vitro pharmacology with EA.hy926 and HUVEC endothelial cells indicated that these ligands are effective binders of αVß3 and potent agonists of PAR2. These were also used in preliminary studies investigating their effects on PAR2 signaling in the presence of inflammatory agents, and represent the first examples of ligands targeting both PARs and integrins, though concurrent binding to αVß3 and PAR2 has not yet been demonstrated.
RESUMEN
The activation state of many blood and vascular cells is tightly controlled by a delicate balance between receptors that contain immunoreceptor tyrosine-based activation motifs (ITAMs) and those that contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Precisely how the timing of cellular activation by ITAM-coupled receptors is regulated by ITIM-containing receptors is, however, poorly understood. Using platelet endothelial cell adhesion molecule 1 (PECAM-1) as a prototypical ITIM-bearing receptor, we demonstrate that initiation of inhibitory signaling occurs via a novel, sequential process in which Src family kinases phosphorylate the C-terminal ITIM, thereby enabling phosphorylation of the N-terminal ITIM of PECAM-1 by other Src homology 2 domain-containing nonreceptor tyrosine kinases (NRTKs). NRTKs capable of mediating the second phosphorylation event include C-terminal Src kinase (Csk) and Bruton's tyrosine kinase (Btk). Btk and Csk function downstream of phosphatidylinositol 3-kinase (PI3K) activation during ITAM-dependent platelet activation. In ITAM-activated platelets that were treated with a PI3K inhibitor, PECAM-1 was phosphorylated but did not bind the tandem SH2 domain-containing tyrosine phosphatase SHP-2, indicating that it was not phosphorylated on its N-terminal ITIM. Csk bound to and phosphorylated PECAM-1 more efficiently than did Btk and required its SH2 domain to perform these functions. Additionally, the phosphorylation of the N-terminal ITIM of Siglec-9 by Csk is enhanced by the prior phosphorylation of its C-terminal ITIM, providing evidence that the ITIMs of other dual ITIM-containing receptors are also sequentially phosphorylated. On the basis of these findings, we propose that sequential ITIM phosphorylation provides a general mechanism for precise temporal control over the recruitment and activation of tandem SH2 domain-containing tyrosine phosphatases that dampen ITAM-dependent signals.
Asunto(s)
Plaquetas/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Transducción de Señal , Agammaglobulinemia Tirosina Quinasa , Secuencias de Aminoácidos , Proteína Tirosina Quinasa CSK , Citoplasma/metabolismo , Humanos , Fosfopéptidos/metabolismo , Fosforilación , Glicoproteínas de Membrana Plaquetaria/metabolismo , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/metabolismo , Dominios Homologos src , Familia-src Quinasas/metabolismoRESUMEN
Immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptors inhibit cellular responsiveness to immunoreceptor tyrosine-based activation motif (ITAM)-linked receptors. Although tyrosine phosphorylation is central to the initiation of both inhibitory ITIM and stimulatory ITAM signaling, the events that regulate receptor phosphorylation are incompletely understood. Previous studies have shown that ITAM tyrosines engage in structure-inducing interactions with the plasma membrane that must be relieved for phosphorylation to occur. Whether ITIM phosphorylation is similarly regulated and the mechanisms responsible for release from plasma membrane interactions to enable phosphorylation, however, have not been defined. PECAM-1 is a dual ITIM-containing receptor that inhibits ITAM-dependent responses in hematopoietic cells. We found that the PECAM-1 cytoplasmic domain is unstructured in an aqueous environment but adopts an α-helical conformation within a localized region on interaction with lipid vesicles that mimic the plasma membrane. The lipid-interacting segment contains the C-terminal ITIM tyrosine and a serine residue that undergo activation-dependent phosphorylation. The N-terminal ITIM is excluded from the lipid-interacting segment, and its phosphorylation is secondary to phosphorylation of the membrane-interacting C-terminal ITIM. On the basis of these findings, we propose a novel model for regulation of inhibitory signaling by ITIM-containing receptors that relies on reversible plasma membrane interactions and sequential ITIM phosphorylation.
Asunto(s)
Membrana Celular/metabolismo , Citoplasma/metabolismo , Fosforilcolina/análogos & derivados , Fosfotirosina/metabolismo , Activación Plaquetaria , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Dicroismo Circular , Humanos , Micelas , Fragmentos de Péptidos/metabolismo , Fosforilación , Fosforilcolina/metabolismo , Agregación Plaquetaria , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/química , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
In mice lacking the blood coagulation regulator thrombomodulin, fibrinolytic degradation products (FDP) of fibrin induce apoptotic cell death of a specialized cell type in the placenta, polyploid trophoblast giant cells. Here, we document that this bioactivity of FDP is conserved in human FDP, is not limited to trophoblast cells, and is associated with an Aalpha-chain segment of fibrin fragment E (FnE). The majority of proapoptotic activity is arginine-glycine-aspartic acid (RGD)-independent and requires caveolin-1-dependent cellular internalization of FnE. Internalization through caveoli is mediated by an epitope contained within Aalpha52-81 that is necessary and sufficient for cellular uptake of FnE. Aalpha52-81 does not cause apoptosis itself, and competitively inhibits FnE internalization and apoptosis induction. Apoptotic activity per se resides within Aalpha17-37 and requires the N-terminal neoepitope generated by release of fibrinopeptide A. Cellular internalization of FnE elicits depression of mitochondrial function and consequent apoptosis that is strictly dependent on the activity of caspases 9 and 3. These findings describe the molecular details of a novel mechanism linking fibrin degradation to cell death in the placenta, which may also contribute to pathologic alterations in nonplacental vascular beds that are associated with fibrinolysis.
Asunto(s)
Apoptosis , Caveolina 1/fisiología , Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Animales , Aorta/citología , Aorta/metabolismo , Caspasa 3/metabolismo , Células Cultivadas , Coriocarcinoma/metabolismo , Coriocarcinoma/patología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Femenino , Glutatión Transferasa/genética , Humanos , Etiquetado Corte-Fin in Situ , Ratones , Ratones Noqueados , Fragmentos de Péptidos/metabolismo , Embarazo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Trofoblastos/citología , Trofoblastos/metabolismo , Venas Umbilicales/citología , Venas Umbilicales/metabolismo , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologíaRESUMEN
The Ig-ITIM family member PECAM-1 is expressed in vascular and endothelial cells, and its functions include suppression of mitochondria-dependent apoptosis. Previous studies have identified distinct PECAM-1 cytoplasmic domain splice variants at the mRNA, but not protein, level. Several relatively abundant mRNA isoforms lack exon 15 (Delta15) and would theoretically encode a protein with a truncated cytoplasmic domain and a unique C-terminal sequence. Using a novel rabbit polyclonal antibody that specifically recognizes Delta15 PECAM-1, we found that the Delta15 PECAM-1 isoform was expressed in human tissues, including brain, testes and ovary. This isoform was also expressed on the cell surface of human platelets, human umbilical vein endothelial cells (HUVECs) and the Jurkat T-cell leukemia, human erythroleukemia (HEL) and U937 histiocytic lymphoma cell lines. Furthermore, murine platelets and lung lysates demonstrated abundant amounts of exon-15-deficient PECAM-1. Functional studies revealed that Delta15 PECAM-1 retains both its homophilic binding capacity and its ability to signal by means of its immunoreceptor tyrosine-based inhibitory motif (ITIM) domains. Delta15 PECAM-1 was unable, however, to protect against apoptosis induced by overexpression of Bax or treatment with the chemotherapy agent etoposide. These studies suggest a novel role for the PECAM-1 C-terminus in cytoprotective signaling and highlight a need for further characterization of expression of PECAM-1 isoforms in normal and malignant tissues.
Asunto(s)
Empalme Alternativo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Transducción de Señal , Secuencia de Aminoácidos , Animales , Apoptosis , Secuencia de Bases , Línea Celular , Cartilla de ADN , Humanos , Ratones , Microscopía Confocal , Datos de Secuencia Molecular , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/química , Homología de Secuencia de AminoácidoRESUMEN
Bax is a proapoptotic protein that plays a key role in the induction of apoptosis. Ku70 has activities to repair DNA damage in the nucleus and to suppress apoptosis by inhibiting Bax in the cytosol. We previously designed peptides based on the amino acid sequence of Bax-binding domain of human Ku70, and showed that these peptides bind Bax and inhibit cell death in human cell lines. In the present report, we examined the biological activities of other pentapeptides, VPTLK and VPALR, derived from mouse and rat Ku70. Cells in culture accumulated FITC-labeled VPTLK and VPALR, indicating that these peptides are cell permeable (human, mouse, rat, and porcine cells were examined). These peptides bound to Bax and suppressed cell death in various cell types including primary cultured cells. These data suggest that such Bax inhibiting peptides from three mammalian species may be used to protect healthy cells from apoptotic injury under pathological conditions.