Mechanisms of u-PAR-Mediated Lung Fibroblast Motility

描述（由申请方提供）：对急性和慢性肺损伤的纤维增生反应导致肺泡破坏和纤维化以及显著的发病率和死亡率。这种反应的特征是间质成纤维细胞迁移到临时肺泡基质中，形成组织病理学可识别的成纤维细胞灶。细胞迁移依赖于蛋白酶尿激酶（u-PAR）的受体，但u-PAR相关的迁移不依赖于尿激酶的蛋白水解活性，u-PAR缺乏跨膜结构域，但与质膜糖基磷脂酰肌醇连接。这些功能限制u-PAR的质膜脂质微域，并建议u-PAR与跨膜受体，包括整合素的相互作用，细胞运动所需的细胞内信号中发挥了重要作用。来自纤维化肺的成纤维细胞在体外和体内表达增加的u-PAR水平，并且u-PAR表达在一些体内模型中改变组织纤维化。这些观察结果导致了这样的假设，即通过u-PAR调节整合素功能影响肺成纤维细胞运动。此外，u-PAR在体外和体内以依赖于u-PAR在脂筏内的限制性定位的方式调节整合素功能。这些假设将在三个具体目标中得到检验。首先，在稳定过表达u-PAR、显示u-PAR下调或阻断u-PAR-整联蛋白相互作用的人肺成纤维细胞中评估粘附和运动性，将从纤维化肺患者分离的成纤维细胞中的u-PAR依赖性运动性与来自非纤维化对照的成纤维细胞进行比较。其次，在稳定过表达野生型u-PAR或非糖基磷脂酰肌醇连接的突变型u-PAR融合蛋白的人肺成纤维细胞中评估粘附和运动性。将在这些成纤维细胞克隆中评估通过粘着斑激酶的运动性信号传导的需求。第三，u-PAR-整联蛋白相互作用和u-PAR的糖基磷脂酰肌醇连接对纤维增生组织浸润的作用将在体内小鼠模型中确定，在该模型中使用特异性抑制肽和基因活化基质实现u-PAR特异性。这项工作将提供新的见解的分子事件，支配纤维增生的过程，并支持设计新的治疗药物，重点是抑制肺损伤的纤维化反应。

DESCRIPTION (provided by applicant): The fibroproliferative response to acute and chronic lung injury results in alveolar destruction and fibrosis and significant morbidity and mortality. This response is characterized by migration of interstitial fibroblasts into a provisional alveolar matrix forming histopathologically recognizable fibroblastic foci. Cellular migration has been shown to depend on the receptor for the protease, urokinase (u-PAR), but u-PAR-related migration is independent of urokinase proteolytic activity, u-PAR lacks a transmembrane domain but is glycosylphosphotidylinositol-linked to the plasma membrane. These features constrain u-PAR to plasma membrane lipid microdomains, and suggest that interactions of u-PAR with transmembrane receptors, including integrins, play a major role in the intracellular signaling necessary for cell motility. Fibroblasts from fibrotic lungs express increased levels of u-PAR in vitro and in vivo, and u-PAR expression alters tissue fibrosis in some in vivo models. These observations have led to the hypothesis that modulation of integrin function by u-PAR affects lung fibroblast motility. Moreover, that u-PAR modulates integrin function in vitro and in vivo in a manner that is dependent on u-PAR's restricted localization within lipid rafts. These hypotheses will be tested in three specific aims. First, adhesion and motility will be assessed in human lung fibroblasts that stably overexpress u-PAR, exhibit downregulation of u-PAR, or have blocked u-PAR-integrin interactions, u-PAR-dependent motility in fibroblasts isolated from patients with fibrotic lungs will be compared with those from non-fibrotic controls. Second, adhesion and motility will be assessed in human lung fibroblasts that stably overexpress either wt u-PAR, or a non-glycosylphosphotidylinositol-linked mutant u-PAR fusion protein. The requirement for motility signaling through focal adhesion kinase will be assessed in these fibroblast clones. Third, the role of u-PAR-integrin interactions and of u-PAR's glycosylphosphotidylinositol link on fibroproliferative tissue infiltration will be determined in an in vivo murine model, u-PAR specificity is accomplished in this model using specific inhibitory peptides and gene-activated matrices. This work will provide new insight into the molecular events which govern the fibroproliferative process, and support the design of novel therapeutic agents focused on inhibiting the fibrotic response to lung injury.