Proteinase Modulation Diring T-Cell Endothelial Adhesion

描述(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一个“蛋白水解性恒温器”，它部分地通过一系列不同的、复杂的传感机制来调节细胞蛋白水解性的表达，其中部分包括选定的基质金属蛋白酶锚定分子(包括MT1-MMP和CD44)和选定的黏附分子(包括免疫球蛋白家族成员PECAM-1)。此外，我们假设这些调节系统影响炎症部位的这些细胞的功能。该项目的目标是： 1.研究CD31(PECAM1)对T淋巴细胞和血管内皮细胞MMPs(MMP2、MMP14、MMP9)表达的调控作用及信号转导途径(S)。我们将确定PECAM1同亲和异亲结合后启动的信号级联(S)。我们将使用WT和PECAM1缺失细胞、PECAM1胞质结构域截断和外显子缺失突变体，并选择Y到F、S到C和S到D的突变体，这些突变体是已知影响PECAM-1-接头/信号分子相互作用的。我们将确定已知参与调节这些MMPs的选定转录因子的水平。 2.阐明细胞表面束缚分子(MT1-MMPs和CD44)对MMP一2和MMP一9表达的调控作用。我们将确定基质金属蛋白酶-2与MT1-基质金属蛋白酶复合体和基质金属蛋白酶-9与CD44结合后的信号通路。我们将检测WT、MMP2缺失、MMP9缺失、MT1缺失和CD44缺失小鼠以及从这些动物分离的内皮细胞和脾细胞中MMP2和MMP9的表达和活性水平。在全长、截短和定点突变的MT1-MMPs和CD44构建体转染后，将对血管内皮细胞和淋巴细胞进行分析。信号通路将使用标准的药理学和遗传学方法来阐明。我们将确定已知参与调节这些MMPs的选定转录因子的水平。 3.阐明MMPs在实验性自身免疫性脑脊髓炎(EAE)中作为趋化因子活性调节剂的作用(S)。我们将评估在体内以及在淋巴细胞和内皮细胞培养中，在EAE发展过程中，基质金属蛋白酶-2、-9和14对特定趋化因子的蛋白分解能力。 4.我们将在我们的EAE抗原特异性炎症的体内小鼠模型中测试从我们的体外数据中得出的结论。具体来说，我们将使用WT、CD31KO、MMP2KO、MMP9KO、MMP14AND-/-和CD44KO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.