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- 2007 (3) (entfernen)
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- Dissertation (3)
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- Englisch (3) (entfernen)
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- Aktin Zytoskelett (1)
- BMP (1)
- Cortical Actin Cytoskeleton (1)
- Cyclo-GMP (1)
- ES-Cells (1)
- Endothelial Cell-Cell Contacts (1)
- Endotheliale Zell-Zell-Kontakte (1)
- Medaka (1)
- Megakaryocytes (1)
- Megakaryozyt (1)
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- Institut für Klinische Biochemie und Pathobiochemie (3) (entfernen)
In physiological conditions platelets have a major role in maintaining haemostasis. Platelets prevent bleeding from wounds by distinguishing normal endothelial cells in vasculature from areas with lesions to which they adhere. Interaction of platelet agonists and their receptors is controlled by intracellular signaling molecules that regulate the activation state of platelets. Very important intracellular signaling molecules are cyclic nucleotides (cGMP and cAMP), both involved in inhibition of platelet activation. Formation of cGMP and cAMP in platelets is stimulated by endothelial-derived NO and prostacyclin (PGI2), which then mediate inhibition of platelets by activating protein kinase G (PKG) and protein kinase A (PKA). Recently, it has been suggested that reactive oxygen species (ROS) represent new modulators of cell signaling within different cell types. The work summarized here describes the involvement of platelet ROS production in platelet activation, the relation of NO/cGMP/PKG I pathway to ROS and to mitogen-activated protein kinases (MAP kinase) signaling, and the involvement of cyclic nucleotides in megakaryocyte and platelet development. Platelets activated with different agonists produce intracellular but not extracellular ROS by activation of NAD(P)H oxidase. In addition, ROS produced in platelets significantly affects αIIbβ3 integrin activation but not alpha/dense granule secretion and platelet shape change. Thrombin induced integrin αIIbβ3 activation is significantly decreased after pretreatment of platelets with NAD(P)H oxidase inhibitors and superoxide scavengers. These inhibitors also reduce platelet aggregation and thrombus formation on collagen under high shear and achieve their effects independently of the NO/cGMP pathway. ADP secreted from platelet dense granules with subsequent activation of P2Y12 receptors as well as thromboxane A2 release are found to be important upstream mediators of p38 MAP kinase activation by thrombin. However, p38 MAP kinase activation does not significantly contribute to calcium mobilization, P-selectin expression, αIIbβ3 integrin activation and aggregation of human platelets in response to thrombin. Finally, PKG activation does not stimulate, but rather inhibit, p38 and ERK MAP kinases in human platelets. Further study revealed that cyclic nucleotides not only inhibit platelet activation, but are also involved, albeit differentially, in megakaryocyte and platelet development. cAMP is engaged in haematopoietic stem cell differentiation to megakaryocytes, and cGMP has no impact on this process. While PKA is already present in stem cells, expression of proteins involved in cGMP signaling (soluble guanylyl cyclase, sGC; PKG) increases with maturation of megakaryocytes. In the final step of megakaryocyte maturation that includes release of platelets, cGMP and cAMP have mild but opposing effects: cGMP increases platelet production while cAMP decreases it indicating a finely regulated process that could depend on stimulus coming from adjacent endothelial cells of sinusoids in bone marrow. The results of this thesis contribute to a better understanding of platelet regulation and of the possible molecular mechanisms involved in megakaryocyte maturation in bone marrow vascular microenvironment.
Directed cortical actin assembly is the driving force for intercellular adhesion. Vasodilator-stimulated phosphoprotein (VASP) participates in actin-fiber formation and VASP activity is regulated by phosphorylations. We screened for endothelial cell proteins, which bind to VASP dependent on its phosphorylation status. Differential proteomics identified αII-spectrin as novel VASP-interacting protein. αII-spectrin binds to the triple GP5-motif in VASP via its SH3 domain. cAMP-dependent protein kinase-mediated VASP phosphorylation at Ser157 inhibits αII-spectrin/VASP complex formation. VASP becomes dephosphorylated upon formation of cell-cell contacts and in confluent but not in sparse endothelial cells αII-spectrin colocalizes with non-phosphorylated VASP at cell-cell junctions. Ectopic expression of the αII-spectrin SH3 domain fused to claudin-5 translocates VASP to cell-cell contacts and is sufficient to initiate the formation of cortical actin cytoskeletons. αII-spectrin SH3 domain overexpression stabilizes cell-cell contacts and decreases endothelial permeability. Conversely, permeability of VASP-deficient endothelial cells is elevated. In a skin edema model, microvascular leakage is increased in VASP-deficient over wild-type mice. We propose that αII-spectrin/VASP complexes regulate cortical actin cytoskeleton assembly with implications for formation of endothelial cell-cell contacts and regulation of vascular permeability.
- 77/83 allerdings inaktiv in Kulturen und Embryonen von Medaka. Dieser Unterschied wird durch Daten aus humanen ES-Zellkulturen unterstützt. Letztere sind ebenfalls komplett STAT3 unabhängig. Die BMP-Smad Kaskade wiederum ist in Medaka-Stammzellen aktiv, Antidifferenzierungsgene wie id2, die durch BMP direkt kontrolliert werden, sind dementsprechend exprimiert. Diese Daten stimmen wiederum mit dem Maussystem überein, während humane ES-Zellen diesbezüglich bislang nicht untersucht wurden. Die Interaktion zwischen verschiedenen Signalwegen ist ein bisher noch nicht gut verstandenes Gebiet. Die Integration verschiedener Signale ist aber speziell für Stammzellen, die ihr Differenzierungsschicksal von winzigen Abweichungen in der Signalmixtur abhängig machen, von entscheidender Bedeutung. Im zweiten Teil der hier vorgelegten Arbeit konnte eine Interaktion zwischen dem BMP-Rezeptor 1a und STAT3 nachgewiesen werden. Diese Interaktion ist offenbar Teil eines variablen Komplexes. Zum ersten Mal war es auch möglich, funktionale Konsequenzen für STAT3 nach Stimulierung des BMP-Rezeptors 1a zu dokumentieren. Nach Belegung des BMP-Rezeptors 1a mit dem mutierten BMP2-A34D wird STAT3 trotz Aktivierung durch Phosphorylierung an Tyrosin 705 im Zytoplasma von Maus Stammzellen festgehalten. Zusammengenommen konnte hier gezeigt werden, dass eine Interaktion zwischen den bislang als isoliert betrachteten Signalwegen BMP-Smad und STAT3 besteht. Des Weiteren wurde das Medaka-Stammzellkultursystem benutzt, um zu zeigen, dass STAT3 für die Pluripotenz von Stammzellen nur im Maussystem eine Rolle spielt, wohingegen BMPZielgene wie id2 in bislang allen getesteten ES-Zellkultursystemen aktiv sind.