ADAM17 - Mediated Shedding in Endothelial Inflammatory Responses

描述(由申请人提供)：在炎症过程中，如动脉粥样硬化，内皮细胞的激活导致控制白细胞募集到损伤部位的膜锚定黏附分子、受体和配体的动态调节。内皮细胞快速调节其细胞表面蛋白质谱的一个潜在的重要机制是通过蛋白水解性“脱落”。除了动态改变细胞表面成分外，脱落还会导致具有不同生物学特性的可溶性胞外结构域的释放。ADAM(去整合素和金属蛋白酶)家族的跨膜蛋白是迄今发现的主要的胞外结构域脱落酶，我们和其他人最近证明，参与白细胞募集和炎症反应的各种蛋白质被ADAM17从细胞表面“脱落”。我们进一步表明，干扰ADAM17介导的内皮细胞或白细胞脱落分别在体外和体内显著抑制白细胞跨内皮细胞的迁移。虽然靶向缺失ADAM17会导致围产期死亡，但我们已经开发出有条件缺失ADAM17的小鼠，这将使我们能够消除内皮细胞中ADAM17的表达，从而直接测试其在体内白细胞募集中的功能。这一建议将：1.进一步表征炎症反应中ADAM17介导的内皮细胞脱落的靶点和生物化学；2.研究依赖于ADAM17介导的脱落的跨内皮细胞迁移的机制，并确定参与这一过程的特定ADAM17底物；3.评估靶向从内皮细胞和白细胞中删除ADAM17对体内炎症反应的影响。这项研究的长期目标是更好地了解控制慢性炎症反应的细胞和分子机制，例如动脉粥样硬化，它仍然是西方世界死亡的主要原因。与公共健康相关：在炎症过程中，如与心血管疾病相关的炎症过程中，血管内皮细胞的激活会导致循环中的白细胞重新进入血管壁，最终可能导致血管堵塞。内皮细胞调节白细胞募集的一个潜在的重要机制是通过蛋白水解性“脱落”来快速调节其细胞表面蛋白储备。除了动态改变细胞表面成分外，脱落还会导致具有不同生物学特性的可溶性胞外结构域的释放。这项提案将重点放在已被公认为蛋白质分解脱落关键酶的蛋白酶ADAM17上。我们已经证明，干扰ADAM17对内皮细胞的脱落足以抑制白细胞在培养的内皮细胞之间的迁移，即“跨内皮迁移”。这项建议将进一步确定和表征ADAM17介导的与炎症反应有关的脱落的靶点。我们还将研究依赖于ADAM17介导的脱落的跨内皮细胞迁移的步骤。最后，我们将通过评估对正常内皮细胞功能的影响，包括对炎症的反应，来研究体内缺失内皮细胞ADAM17的影响。我们提案的总体目标是测试ADAM17在炎症反应中充当守门人的假设。这项研究的长期目标是更好地了解控制慢性炎症反应的细胞和分子机制，例如动脉粥样硬化，动脉粥样硬化仍然是西方世界的主要死亡原因。

DESCRIPTION (provided by applicant): Activation of endothelial cells during inflammatory processes such as atherosclerosis results in dynamic regulation of membrane-anchored adhesion molecules, receptors and ligands that control leukocyte recruitment to sites of injury. One potentially important mechanism for endothelial cells to rapidly modulate their repertoire of cell surface proteins is through proteolytic "shedding". In addition to dynamically altering the cell surface constituents, shedding also leads to the release of soluble ectodomains with distinct biological properties. The ADAM (for A Disintegrin And Metalloproteinase) family of transmembrane proteins are the major ectodomain sheddases identified to date, and we and others have recently demonstrated that a wide variety of proteins involved in leukocyte recruitment and inflammatory responses are `shed' from the cell surface by ADAM17. We have further shown that interference with ADAM17-mediated endothelial or leukocyte shedding significantly inhibits leukocyte transendothelial cell migration in vitro and in vivo, respectively. Although targeted deletion of ADAM17 leads to perinatal lethality, we have developed mice with conditional deletion of ADAM17 that will allow us to eliminate ADAM17 expression from endothelial cells and thus directly test its function in leukocyte recruitment in vivo. This proposal will: 1. further characterize targets and biochemistry of ADAM17-mediated shedding in the endothelium during inflammatory responses; 2. Examine the mechanisms involved in transendothelial cell migration dependent upon ADAM17-mediated shedding, and to identify the specific ADAM17 substrates that are contributing to the process; 3. Evaluate the impact of targeted deletion of ADAM17 from endothelial cells and leukocytes on inflammatory responses in vivo. The long-term objective of this research is to better understand the cellular and molecular mechanisms controlling chronic inflammatory responses, such as atherosclerosis, that remains the major cause of death in the Western world. PUBLIC HEALTH RELEVANCE: Activation of the endothelial cells that line blood vessels during inflammatory processes such as those associated with cardiovascular disease results in the recruitment of circulating white blood cells into the vessel wall that can ultimately contribute to blockage of the blood vessel. One potentially important mechanism for endothelial cells to regulate the recruitment of white blood cells is to rapidly modulate their repertoire of cell surface proteins through proteolytic "shedding". In addition to dynamically altering the cell surface constituents, shedding also leads to the release of soluble ectodomains with distinct biological properties. This proposal will focus on the protease ADAM17 that has gained recognition as a key enzyme in proteolytic shedding. We have shown that interference with ADAM17 shedding on endothelial cells is sufficient to inhibit white blood cell migration across cultured endothelial cells, "transendothelial migration". This proposal will further identify and characterize targets of ADAM17-mediated shedding involved in inflammatory responses. We will also examine the steps in transendothelial cell migration that are dependent upon ADAM17-mediated shedding. Finally, we will investigate the impact of in vivo deletion of endothelial cell ADAM17 by evaluating effects on normal endothelial cell functions, including the response to inflammation. The overall goal of our proposal is to test the hypothesis that ADAM17 serves as a gatekeeper during inflammatory responses. The long-term objective of this research is to better understand the cellular and molecular mechanisms controlling chronic inflammatory responses such as atherosclerosis that remains the major cause of death in the Western world.