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: 10229371
• 负责人: SANFORD J SHATTIL
• 金额: $ 50.17万
• 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/10229371
• 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; Gap Junctions; 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; Lung; MHC Class I Genes; Mediating; Megakaryocytes; Modeling; Mus; NF-kappa B; 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; base; cadherin 5; conditional knockout; in vivo; induced pluripotent stem cell; insight; interest; knock-down; 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.

项目总结 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起重要作用。 止血。纤维蛋白原与血小板的结合受特定细胞内蛋白的相互作用调节，包括 Talin和Kindlin-3，带有β-3胞质尾巴。相比之下，可能与αIIb尾巴相互作用的蛋白质 纤维蛋白原结合的研究相对较少。我们发现人类和小鼠的血小板和内皮细胞 细胞表达40 kDa的蛋白质夏尔平。对人类血小板的研究以及对血小板和 人类诱导多能干细胞来源的巨核细胞显示夏平可以相互作用 直接与αIIb尾部或与另外两种蛋白质共同构成线性泛素化链组装体 复合体(LUBAC)。事实上，传统的止血激动剂，如凝血酶，或通过 炎症激动剂，如脂多糖或可溶性CD40配体(SCD40L)，触发这两种纤维蛋白原 结合αIIbβ3和MET1连接的IKKγ的线性泛素化(NEMO)促进NF-kB通路信号转导。 巨核细胞和血小板中shRNA敲除Sharpin导致激动剂诱导的线性 NEMO的泛素化，但增加了纤维蛋白原与αIIbβ3的结合，MHC I类的表达和释放 内源性sCD40L。在这里，我们将检验这样一种假设，即夏平与 整合素α尾或LUBAC在止血过程中调节关键的血小板和/或内皮细胞反应， 血栓形成、炎症和血管生成。目标1号将使用包括光遗传学在内的先进技术来 测定血小板夏平和αIIbβ3的化学计量比，并检测强迫剂对其功能的影响 锐化与αIIb或LUBAC的交互。将产生血小板特异性Sharpin基因敲除小鼠 为了测试止血、血栓形成和炎症中对血小板夏普林的需求，使用一系列 老鼠模型。目标2将确定Sharpin在整合素的黏附和血管生成功能中的作用 αV、β3及在内皮细胞中的核因子-kB信号转导。内皮细胞夏平将特异性地和 在小鼠中条件敲除，这些小鼠的肺微血管内皮细胞将被评估 αVβ3依赖的黏附反应和血管生成发芽。去除内皮细胞的效果 将利用已建立的小鼠发育和病理模型来确定体内的夏尔平 血管生成。该项目将大量使用止血、血栓和炎症模型核心 它将与该计划中的所有其他项目合作，以实现其目标。总而言之，这些研究将 对中心假说进行全面检验，并建立对监管的新的机械论见解 血管细胞中整合素和夏尔平免疫信号的研究，具有明显的止血作用， 血栓形成、炎症和血管生成。