LOCALIZATION AND ACTIVATION OF METALLOPROTEINASES

白细胞来源的基质金属蛋白酶(MMPs)对细胞外基质成分的损伤是肺气肿、囊性纤维化、急性呼吸窘迫综合征和类风湿性关节炎等慢性疾病致残的关键致病事件。基质金属蛋白酶介导的组织损伤的流行概念是基质金属蛋白酶以酶原的形式自由分泌到细胞外空间。然而，关于proMMPs在体内被激活的机制，或者它们如何避开细胞外空间内高亲和力抑制剂的影响，现有的信息很少。我们的初步数据表明，人白细胞表面表达有基质溶素、中性粒细胞胶原酶和明胶酶9(92 kDa明胶酶、92 kDa IV型胶原酶和明胶酶B)，并且它们的表达受促炎介质的上调。我们建议检验这一假设，即当MMPs被限制在白细胞的细胞表面时，它们被聚焦并受到保护，不受自然产生的抑制剂的影响。为此，我们将追求以下特定的目标：1.确定哪些MMPs(MMP7、MMP8、MMP9和MMP12)在中性粒细胞和单核细胞的细胞表面表达，定量它们在细胞激活时的表达，并探讨MMPs和激活的细胞膜上的黏附分子协同表达促进MMP介导胞外蛋白分解的可能性。2.研究MMPs与炎症细胞表面结合对酶原激活、催化活性和抑制敏感性的影响。3.探讨MMPs与白细胞膜结合的机制。我们期望所提出的研究将为MMPs介导组织损伤的机制提供新的见解，并且这些知识将促进许多慢性和致残性疾病的有效治疗策略的合理发展，在这些疾病中，MMPs介导的组织损伤是关键事件。

Injury to extracellular matrix components by matrix metalloproteinases (MMPs) derived from leukocytes is a pivotal pathogenetic event in disabling chronic diseases such as pulmonary emphysema, cystic fibrosis, acute respiratory distress syndrome, and rheumatoid arthritis. The prevailing concept of MMP-mediated tissue injury is that MMPs are freely secreted into the extracellular space as proenzymes. However, there is little information available about the mechanisms by which proMMPs are activated in vivo, or how they circumvent the effects of high- affinity inhibitors within the extracellular space. Our preliminary data indicate that MMP-7 (matrilysin), MMP-8 (neutrophil collagenase), and MMP-9 (92 kDa gelatinase; 92 kDa type IV collagenase; gelatinase B) are expressed on the cell surface of human leukocytes, and that their expression is upregulated by pro-inflammatory mediators. We propose to test the hypothesis that when MMPs are confined to the cell surface of leukocytes, they are focused and protected from naturally- occurring inhibitors. In doing so, we will pursue the following Specific Aims: 1. Determine which MMPs (MMP-7, -8, -9 and MMP- 12[macrophage metalloelastase]) are expressed on the cell surface of neutrophils and monocytes, quantify their expression in response to cellular activation, and investigate the possibility that coordinate expression of MMPs and adhesion molecules on activated cell membranes facilitates MMP-mediated extracellular proteolysis. 2. Examine the consequences of binding of MMPs to the cell surface of inflammatory cells with respect to proenzyme activation, catalytic activity and susceptibility to inhibition. 3. Investigate the mechanisms of binding of MMPs to leukocyte cell membranes. We anticipate that the proposed studies will provide novel insights into the mechanisms by which MMPs mediate tissue injury, and that this knowledge will facilitate rational development of effective therapeutic strategies for many chronic and disabling diseases in which MMP-mediated tissue injury is a critical event.