Role of SHARPIN in the Adhesive and Inflammatory Functions of Platelets and Endothelial Cells

SHARPIN 在血小板和内皮细胞粘附和炎症功能中的作用

• 批准号: 10676905
• 负责人: SANFORD J SHATTIL
• 金额: $ 49.78万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-05 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676905
• 关键词: Adaptor Signaling Protein; Adhesives; Agonist; Binding; Blood Platelets; Blood Vessels; CD40 Ligand; Cell physiology; Cells; Collaborations; Complex; Cytoplasmic Tail; Development; Endothelial Cells; Fibrinogen; Fibrinogen Receptors; Hemostatic Agents; Hemostatic function; Human; Immune; Immune signaling; Immunity; Inflammation; Inflammatory; Integrin alpha Chains; Integrin alphaVbeta3; Integrins; Knock-out; Knockout Mice; Lentivirus; Link; Lipopolysaccharides; LoxP-flanked allele; MHC Class I Genes; Mediating; Megakaryocytes; Modeling; Mus; NFKB Activation Pathway; NFKB Signaling Pathway; Partner in relationship; Pathologic Neovascularization; Pathway interactions; Platelet Membrane Glycoprotein IIb; Platelet aggregation; Proteins; Regulation; Research; Role; Signal Pathway; Signal Transduction; Tail; Talin; Techniques; Testing; Thrombin; Thrombosis; Tumor Angiogenesis; Ubiquitin; Ubiquitination; Western Blotting; angiogenesis; cadherin 5; conditional knockout; in vivo; induced pluripotent stem cell; insight; interest; knock-down; lung microvascular endothelial cells; mouse model; optogenetics; overexpression; platelet function; preservation; programs; response; small hairpin RNA; stoichiometry; tool; ubiquitin-protein ligase

PROJECT SUMMARY Integrin αIIbβ3 (GP IIb-IIIa) is the platelet receptor for fibrinogen and is required for platelet aggregation during hemostasis. Fibrinogen binding to platelets is regulated by interactions of specific intracellular proteins, including talin and kindlin-3, with the β3 cytoplasmic tail. In contrast, proteins that might interact with the αIIb tail to regulate fibrinogen binding are relatively unexplored. We have found that human and mouse platelets and endothelial cells express the 40 kDa protein, SHARPIN. Studies with human platelets as well as with platelets and megakaryocytes derived from human induced pluripotent stem cells have revealed that SHARPIN can interact directly with either the αIIb tail or with two other proteins to constitute the linear ubiquitination chain assembly complex (LUBAC). In fact, stimulation of platelets by traditional hemostatic agonists, such as thrombin, or by inflammatory agonists, such as lipopolysaccharide or soluble CD40 ligand (sCD40L), triggers both fibrinogen binding to αIIbβ3 and Met1-linked linear ubiquitination of IKKγ (NEMO) to promote NF-kB pathway signaling. SHARPIN knockdown by shRNA in megakaryocytes and platelets results in decreased agonist-induced, linear ubiquitination of NEMO, but increased fibrinogen binding to αIIbβ3, MHC Class I expression, and release of endogenous sCD40L. Here we will test the hypothesis that SHARPIN’s mutually exclusive interactions with integrin α tails or LUBAC regulate critical platelet and/or endothelial cell responses during hemostasis, thrombosis, inflammation and angiogenesis. Aim 1 will use advanced techniques, including optogenetics, to determine the stoichiometry of SHARPIN and αIIbβ3 in platelets and to test the functional effects of enforcing SHARPIN interactions with either αIIb or LUBAC. Platelet-specific SHARPIN knockout mice will be generated in order to test the requirement for platelet SHARPIN in hemostasis, thrombosis and inflammation using a range of mouse models. Aim 2 will determine the role of SHARPIN in the adhesive and angiogenic functions of integrin αVβ3 and in NF-kB pathway signaling in endothelial cells. Endothelial cell SHARPIN will be specifically and conditionally knocked out in mice, and lung microvascular endothelial cells from these mice will be evaluated for αVβ3-dependent adhesive responses and for angiogenic sprouting. The effects of deleting endothelial cell SHARPIN in vivo will be determined using established mouse models of developmental and pathological angiogenesis. This project will make heavy use of the Hemostasis, Thrombosis, and Inflammation Models Core and it will collaborate with all other projects in this Program to achieve its aims. Altogether, these studies will provide a comprehensive test of the central hypothesis and establish new mechanistic insights into the regulation of integrin and immune signaling by SHARPIN in vascular cells, with clear implications for hemostasis, thrombosis, inflammation and angiogenesis.

项目摘要 整合素αIIbβ3（GP IIb-IIIa）是纤维蛋白原的血小板受体，在血小板聚集过程中是必需的。 止血。纤维蛋白原与血小板的结合受特定细胞内蛋白质相互作用的调节，包括 talin和kindlin-3，具有β3胞质尾。相反，可能与αIIb尾部相互作用以调节 纤维蛋白原结合是相对未探索的。我们发现人和小鼠的血小板和内皮细胞 细胞表达40 kDa蛋白SHARPIN。使用人血小板以及血小板和 来自人类诱导多能干细胞的巨核细胞揭示了SHARPIN可以与 直接与αIIb尾或与另外两种蛋白质一起构成线性泛素化链组装体 综合体（LUBAC）。事实上，通过传统的止血激动剂如凝血酶，或通过抗凝血剂对血小板的刺激， 炎症激动剂，如脂多糖或可溶性CD 40配体（sCD 40 L）， 结合αIIbβ3和Met 1连接的IKKγ线性泛素化（NEMO）以促进NF-κ B通路信号传导。 在巨核细胞和血小板中通过shRNA敲低SHARPIN导致激动剂诱导的线性 NEMO的泛素化，但增加纤维蛋白原与αIIbβ3的结合，MHC I类表达， 内源性sCD 40 L。在这里，我们将测试的假设，SHARPIN的相互排斥的相互作用， 整联蛋白α尾或LUBAC调节止血期间关键血小板和/或内皮细胞应答， 血栓形成、炎症和血管生成。Aim 1将使用包括光遗传学在内的先进技术， 测定血小板中SHARPIN和αIIbβ3的化学计量，并测试增强的功能效应。 SHARPIN与αIIb或LUBAC的相互作用。血小板特异性SHARPIN敲除小鼠将在 为了测试血小板SHARPIN在止血、血栓形成和炎症中的需求，使用一系列 小鼠模型。目的2：探讨SHARPIN在整合素粘附和血管生成中的作用 αVβ3和NF-κ B信号通路在内皮细胞中的作用。内皮细胞SHARPIN将特异性地和 在小鼠中条件性敲除肺微血管内皮细胞，并且将评价来自这些小鼠的肺微血管内皮细胞的功能。 αVβ3依赖性粘附反应和血管生成发芽。去除内皮细胞的影响 SHARPIN在体内将使用已建立的发育和病理学的小鼠模型来确定。 血管生成本项目将大量使用止血、血栓形成和炎症模型核心 并将与该计划中的所有其他项目合作，以实现其目标。这些研究将 提供了一个全面的测试中心假设，并建立新的机制的监管见解 SHARPIN在血管细胞中的整合素和免疫信号传导，对止血有明确的意义， 血栓形成、炎症和血管生成。