E-SELECTIN DEPENDENT COMMUNICATION IN INFLAMMATION

E-选择素(ELAM-1，CD62E)，一种细胞因子诱导的内皮特异性 黏附受体选择素家族的成员，支持滚动 并稳定地阻止被激活的表面选定的血白细胞 体外和体内的血管内皮细胞。最近的数据表明，在 除了这种支持黏附的功能外，E-选择素还 在向内皮细胞传递信号的过程中发挥作用，以及 在生物力学活动中，如细胞骨架锚定。在这个持续的过程中 项目中，我们将检验E-选择素分子介导 白细胞-内皮细胞间和细胞内通讯 炎症部位的粘连相互作用。 在具体目标1中，形成的动力学和地形，以及 “顶端焦点粘连复合体(顶端)”的生化成分 由E-选择素依赖的白细胞与 激活的内皮细胞，将使用组合 共聚焦免疫荧光显微镜、免疫电子显微镜和 免疫化学。 在具体目标2中，调节磷酸化的机制/ 不同条件下E-选择素胞浆结构域去磷酸化的研究 将检查白细胞-内皮相互作用的条件； 特定氨基酸残基的参与将通过以下方式确定 突变；细胞骨架结合的功能意义， 将探索心尖部FAC的形成和细胞内信号事件。 在特定的目标#3中，E-选择素的“下游”分子靶标-- 将研究内皮细胞中的中介信号， 包括：丝裂原激活蛋白激酶(MAP-K)的激活； 血管内皮细胞表达的黏附分子和细胞因子 炎症反应与内皮细胞E-选择素的自身调节 通过E-选择素转导信号表达。 在特定的目标4中，E-选择素的病理生理后果- 相关的细胞间和细胞内信号将在 各种体外和体内白细胞-内皮相互作用的模型， 利用开发的独特的细胞和分子生物试剂 在本项目中，包括E-选择素缺陷小鼠，培养 来源于这些动物的微血管内皮细胞和腺病毒 野生型和突变型高效可滴定表达载体 E-选择素分子。 这些研究的结果应该会增加我们对 血管内皮细胞在损伤和炎症反应中的积极作用 反应，特别是E-选择的潜在贡献 在这三个过程中细胞间和细胞内的通讯。

E-Selectin (ELAM-1, CD62E), a cytokine-inducible, endothelial-specific member of the Selectin family of adhesion receptors, supports the rolling and stable arrest of selected blood leukocytes on the surface of activated vascular endothelium in vitro and in vivo. Recent data indicate that, in addition to this adhesion-supporting function, E-selectin also is functioning in the transduction of signals into the endothelial cells, and in biomechanical events such as cytoskeletal anchoring. In this continuing project, we will test the hypothesis that the E-selectin molecule mediates intercellular and intracellular communication during leukocyte-endothelial adhesive interactions at sites of inflammation. In Specific Aim #1, the kinetics and topography of formation, and biochemical composition, of "apical focal adhesion complexes (apical FACs)", induced by E-selectin-dependent adhesion of leukocytes to activated endothelial cells, will be characterized using a combination of confocal immunofluorescence microscopy, immuno-electron microscopy and immunochemistry. In Specific Aim #2, the mechanisms that regulate the phosphorylation/ dephosphorylation of the cytoplasmic domain of E-selectin under various conditions of leukocyte-endothelial interaction will be examined; the involvement of specific amino acid residues will be determined by mutagenesis; and the functional significance for cytoskeletal association, apical FAC formation and intracellular signaling events will be explored. In Specific Aim #3, a "downstream" molecular targets of E-selectin- mediated signaling in the endothelial cell will be investigated, including: mitogen-activate protein kinase (MAP-kinase) activation; lateral endothelial-expressed adhesion molecules and cytokines relevant to the inflammatory response; and autoregulation of endothelial E-selectin expression by E-selectin-transduced signals. In Specific AIM #4, the pathophysiologic consequences of E-selectin- dependent intercellular and intracellular signaling will be examined in various in vitro and in vivo models of leukocyte-endothelial interaction, making use of unique cellular and molecular biological reagents developed in this project, including E-selectin-deficient mice, cultured microvascular endothelial cells derived from these animals, and adenoviral vectors for the efficient and tiratable expression of wildtype and mutant E-selectin molecules. The results of these studies should increase our understanding of the active role of vascular endothelium in response-to-injury and inflammatory reactions and, in particular, the potential contribute of E-selecting to intercellular and intracellular communication during thee processes.