Mechanisms of u-PAR-Mediated Lung Fibroblast Motility

描述（由申请人提供）：急性和慢性肺损伤的纤维增生性反应导致肺泡破坏和纤维化，发病率和死亡率很高。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，形成组织病理学上可识别的成纤维细胞灶。细胞迁移已被证明依赖于蛋白酶受体尿激酶（u-PAR），但u-PAR相关的迁移独立于尿激酶蛋白水解活性，u-PAR缺乏跨膜结构域，但糖基磷脂酰肌醇与质膜相连。这些特征将u-PAR限制在质膜脂微域，并表明u-PAR与跨膜受体（包括整合素）的相互作用在细胞运动所需的细胞内信号传导中起主要作用。在体外和体内，来自纤维化肺的成纤维细胞表达水平升高的u-PAR，在一些体内模型中，u-PAR表达改变了组织纤维化。这些观察结果导致了u-PAR调节整合素功能影响肺成纤维细胞运动的假设。此外，u-PAR在体外和体内调节整合素功能的方式依赖于u-PAR在脂筏中的受限定位。这些假设将在三个具体目标中得到检验。首先，将对稳定过表达u-PAR、u-PAR下调或u-PAR-整合素相互作用被阻断的人肺成纤维细胞的粘附性和运动能力进行评估，将从纤维化肺患者分离的成纤维细胞的u-PAR依赖性运动能力与非纤维化对照组的成纤维细胞进行比较。其次，将对稳定过表达wt - par或非糖基磷脂酰肌醇连接突变型u-PAR融合蛋白的人肺成纤维细胞的粘附性和运动性进行评估。我们将在这些成纤维细胞克隆中评估通过黏附激酶传递运动信号的需求。第三，将在体内小鼠模型中确定u-PAR-整合素相互作用和u-PAR的糖基磷脂酰肌醇连接在纤维增生性组织浸润中的作用，在该模型中使用特异性抑制肽和基因激活基质实现u-PAR特异性。这项工作将为控制纤维增殖过程的分子事件提供新的见解，并支持设计专注于抑制肺损伤纤维化反应的新型治疗剂。

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.