Proteinase Modulation Diring T-Cell Endothelial Adhesion

描述（由申请人提供）：我们的假设是，淋巴细胞、中性粒细胞和内皮细胞表现出“蛋白水解恒温器”，其部分地通过一系列多样化、复杂的传感机制调节细胞蛋白水解活性的表达，这些机制部分地由选定的MMP束缚分子（包括MT1-MMP和CD44）和选定的粘附分子（包括Ig家族成员PECAM-1）组成。此外，我们假设这些调节系统影响炎症部位这些细胞的功能。该项目的目标是： 1. 表征 T 淋巴细胞和内皮细胞中 CD31 (PECAM-1) 介导的 MMP（MMP-2、MMP-14 [MT1-MMP] 和 MMP-9）诱导调节所涉及的信号级联。我们将确定 PECAM-1 嗜同性和异嗜性参与后启动的信号级联。我们将使用 WT 和 PECAM-1 无效细胞、PECAM-1 胞质结构域截短和外显子缺失突变体，以及已知影响 PECAM-1 接头/信号分子相互作用的选定 Y 至 F、S 至 C 和 S 至 D 突变体。我们将确定已知参与调节这些 MMP 的选定转录因子的水平。 2. 阐明细胞表面束缚分子（MT1-MMP 和 CD44）作为 MMP-2 和 MMP-9 表达调节剂的作用。我们将确定 MMP-2 与 MT1-MMP 复合物以及 MMP-9 与 CD44 结合后的信号传导途径。我们将测定 WT、MMP-2 null、MMP-9 null、MT1-MMP & null 和 CD44 null 小鼠以及从这些动物中分离的内皮细胞和脾细胞中的 MMP-2 和 MMP-9 表达和活性水平。在用全长、截短和位点突变的 MT1-MMP 和 CD 44 构建体转染后，将对内皮细胞和淋巴细胞进行分析。将使用标准药理学和遗传学方法阐明信号通路。我们将确定已知参与调节这些 MMP 的选定转录因子的水平。 3. 阐明 MMP 作为实验性自身免疫性脑脊髓炎 (EAE) 中选定趋化因子活性调节剂的作用。我们将评估 MMP-2、-9 和 14 在体内以及淋巴细胞和内皮细胞培养物中 EAE 发生过程中蛋白水解选定趋化因子的能力。 4. 我们将在 EAE 抗原特异性炎症的体内小鼠模型中测试从体外数据得出的结论。具体来说，我们将使用WT、CD31 KO、MMP-2 KO、MMP-9 KO、MMP-14和-/-和CD44 KO小鼠，以及从这些小鼠产生的骨髓嵌合小鼠。

DESCRIPTION (provided by applicant): Our hypothesis is that lymphocytes, PMNs and endothelial cells exhibit a "proteolytic thermostat" which, in part, regulates the expression of cellular proteolytic activity via a series of diverse, complex sensing mechanisms comprised, in part, of selected MMP tethering molecules including MT1-MMP and CD44 and selected adhesion molecules including the Ig family member PECAM-1. In addition we postulate that these regulatory systems affect the functions of these cells at sites of inflammation. The goals of this project are to: 1. Characterize the signaling cascade(s) involved in the CD31 (PECAM-1)-mediated modulation of the induction of MMPs (MMP-2, MMP-14 [MT1-MMP] and MMP-9) in T lymphocytes and endothelial cells. We will determine the signaling cascade(s) initiated following PECAM-1 homophilic and heterophilic engagement. We will use WT and PECAM-1 null cells, PECAM-1 cytoplasmic domain truncation and exon deletion mutants and selected Y to F and S to C and S to D mutants known to affect PECAM-1-adaptor/ signaling molecule interactions. We will determine the levels of selected transcription factors known to be involved in modulating these MMPs. 2. Elucidate the roles of cell surface tethering molecules (MT1-MMP and CD44) as modulators of MMP-2 and MMP-9 expression. We will determine the signaling pathways following tethering of MMP-2 to the MT1- MMP complex and MMP-9 to CD44. We will determine MMP-2 and MMP-9 expression and activity levels in WT, MMP-2 null, MMP-9 null, MT1-MMP & null and CD44 null mice and endothelial cells and splenocytes isolated from these animals. Analyses will be performed on endothelial cells and lymphocytes following transfection with full-length, truncated and site-mutagenized MT1-MMP and CD 44 constructs. Signaling pathways will be elucidated using standard pharmacological and genetic approaches. We will determine the levels of selected transcription factors known to be involved in modulating these MMPs. 3. Elucidate the role(s) of MMPs as modulators of selected chemokine activities in experimental autoimmune encephalomyelitis (EAE). We will assess the abilities of MMP-2, -9 and 14 to proteolyze selected chemokines during the development of EAE in vivo and in lymphocyte and endothelial cell cultures. 4. We will test conclusions drawn from our in vitro data in our in vivo murine model of antigen-specific inflammation in, EAE. Specifically we will use WT, CD31 KO, MMP-2 KO, MMP-9 KO, MMP-14 and -/- and CD44 KO mice, and bone marrow chimeric mice generated from these mice.