Mechanisms of u-PAR-Mediated Lung Fibroblast Motility

描述(由申请人提供)：对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，以及显著的发病率和死亡率。这种反应的特点是间质成纤维细胞迁移到临时的肺泡基质中，形成组织病理学上可识别的成纤维细胞灶。细胞迁移依赖于蛋白水解酶受体(u-PAR)，但与u-PAR相关的迁移不依赖于尿激酶的蛋白分解活性，u-PAR缺乏跨膜区，但糖基化磷脂酰肌醇连接到质膜上。这些特征将u-PAR限制在质膜脂质微区，并提示u-PAR与包括整合素在内的跨膜受体的相互作用在细胞运动所需的细胞内信号转导中发挥重要作用。来自纤维化肺的成纤维细胞在体外和体内表达高水平的u-PAR，在一些体内模型中，u-PAR的表达改变了组织纤维化。这些观察结果导致了一种假设，即u-PAR对整合素功能的调节会影响肺成纤维细胞的运动。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在脂筏中的限制性定位。这些假设将在三个具体目标上得到检验。首先，将评估稳定高表达u-PAR、u-PAR下调或阻断u-PAR-整合素相互作用的人肺成纤维细胞的黏附和运动能力，并将纤维化肺患者分离的成纤维细胞的u-PAR依赖的运动能力与非纤维化对照的细胞进行比较。其次，将评估稳定过表达wt u-PAR或非糖基磷酸肌醇连接的突变u-PAR融合蛋白的人肺成纤维细胞的粘附性和运动性。将在这些成纤维细胞克隆中评估通过粘着斑激酶传递运动信号的需求。第三，u-PAR-整合素的相互作用和u-PAR的糖基磷酸肌醇连接在小鼠体内纤维组织渗透中的作用将被确定，u-PAR的特异性在这个模型中是使用特定的抑制肽和基因激活的基质来完成的。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，并支持以抑制肺损伤的纤维化反应为重点的新型治疗药物的设计。

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.