E-SELECTIN DEPENDENT COMMUNICATION IN INFLAMMATION

E-选择素（ELAM-1，CD 62 E），一种可诱导的内皮特异性 粘附受体的选择蛋白家族的成员，支持滚动 和稳定的逮捕选定的血液白细胞的表面上的活化 体外和体内血管内皮。最近的数据显示，在 除了这种粘附支持功能外，E-选择素还 在将信号转导到内皮细胞中起作用，和 在生物力学事件中，如细胞骨架锚定。在这场持续的 项目，我们将测试的假设，E-选择素分子介导的 白细胞-内皮细胞间和细胞内通讯 炎症部位的粘附相互作用。 在具体目标#1中，形成的动力学和地形，以及 生物化学组成，“顶端粘着斑复合物（顶端 FACs）"，由E-选择素依赖性白细胞粘附至 活化的内皮细胞，将使用以下组合来表征： 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/ E-选择素的胞质结构域在各种条件下的去磷酸化 将检查白细胞-内皮细胞相互作用的条件; 具体氨基酸残基的参与将通过 诱变;以及细胞骨架结合的功能意义， 将探索顶端FAC形成和细胞内信号传导事件。 在特定目标#3中，E-选择素的“下游”分子靶向- 将研究内皮细胞中介导的信号传导， 包括：丝裂原活化蛋白激酶（MAP-激酶）的活化; 内皮细胞表达的粘附分子和细胞因子 炎症反应和内皮细胞E-选择素的自动调节 通过E-选择素转导的信号表达。 在特定的AIM #4中，E-选择素的病理生理后果- 依赖的细胞间和细胞内信号传导将在 白细胞-内皮相互作用的各种体外和体内模型， 利用开发的独特的细胞和分子生物学试剂 在这个项目中，包括E-选择素缺陷小鼠，培养 来自这些动物的微血管内皮细胞，和腺病毒 用于有效和可滴定地表达野生型和突变体的载体 E-选择素分子 这些研究的结果应该增加我们对 血管内皮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， 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.