ADAM17 - Mediated Shedding in Endothelial Inflammatory Responses

描述（由申请人提供）：炎症过程（如动脉粥样硬化）中内皮细胞的激活导致膜锚定粘附分子、受体和配体的动态调节，这些分子、受体和配体控制白细胞招募到损伤部位。内皮细胞快速调节其细胞表面蛋白库的一个潜在的重要机制是通过蛋白水解“脱落”。除了动态改变细胞表面成分外，脱落还导致具有不同生物学特性的可溶性外结构域的释放。ADAM （A Disintegrin And Metalloproteinase）跨膜蛋白家族是迄今为止发现的主要的外膜结构域脱落酶，我们和其他人最近已经证明，参与白细胞募集和炎症反应的各种蛋白都被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迁移。虽然靶向缺失ADAM17会导致围产期死亡，但我们已经培养出条件缺失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。本提案将：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特征；2. 研究依赖于ADAM17介导的脱落的跨内皮细胞迁移的机制，并确定在这一过程中起作用的特定ADAM17底物；3. 评估内皮细胞和白细胞靶向删除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，如动脉粥样硬化，这仍然是西方世界死亡的主要原因。公共卫生相关性：在炎症过程（如与心血管疾病相关的炎症过程）中，血管内皮细胞的活化导致循环白细胞聚集到血管壁上，最终导致血管阻塞。内皮细胞调节白细胞募集的一个潜在重要机制是通过蛋白水解“脱落”快速调节其细胞表面蛋白库。除了动态改变细胞表面成分外，脱落还导致具有不同生物学特性的可溶性外结构域的释放。这一建议将重点放在蛋白酶ADAM17上，该酶已被认为是蛋白水解脱落的关键酶。我们已经证明，干扰内皮细胞上ADAM17的脱落足以抑制白细胞在培养的内皮细胞间的迁移，即“跨内皮迁移”。该提案将进一步确定和表征参与炎症反应的adam17介导的脱落靶点。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，来研究内皮细胞ADAM17在体内缺失的影响。我们提案的总体目标是验证ADAM17在炎症反应中充当看门人的假设。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，如动脉粥样硬化，这仍然是西方世界死亡的主要原因。

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.