KGF and Protection from Hyperoxic Lung Injury

描述(申请人提供)：高氧性肺损伤与广泛的上皮和内皮损伤和细胞死亡以及呼吸道炎症、水肿和出血有关。利用可诱导的肺特异性转基因系统，我们在体内和体外都证明了角质形成细胞生长因子(KGF)对肺上皮细胞具有深刻的保护作用，并激活了促生存的Akt途径。我们已经确定了细胞内的分子，如PAK4和p90RSK，它们与KGF受体(KGFR)相互作用，并在肺上皮细胞中诱导促生存机制。我们还发现在哺乳动物细胞中PAK4与Raf-1相互作用。最近研究表明，RAF-1在细胞存活过程中起着关键作用。我们发现KGF的另一个特性是阻止中性粒细胞迁移到肺泡腔，这是氧化损伤过程中的另一个有害事件。在相关研究中，我们发现晚期糖基化终产物受体(SRAGE)的可溶性形式阻止了中性粒细胞在高氧条件下进入肺内。这些观察结果使我们假设KGF和SRAGE通过影响细胞存活和抗炎机制来抑制氧化性肺损伤。为了验证这一假说，我们将：目的1.确定由激活的KGFR诱导的信号网络在上皮细胞对高氧反应中的功能重要性。A)小鼠肺上皮细胞(MLE-12)在存在或不存在KGF和KGFR相互作用蛋白显性-阴性突变体的情况下，将受到高氧的影响。对a)促和抗凋亡蛋白的亚细胞分布和修饰以及b)Akt和BAD磷酸化的影响将随之而来。目的II.研究KGFR相互作用分子在Akt和体内不良的磷酸化和上皮保护中的作用，a)在可诱导的KGF转基因小鼠背景下，c-Raf-1或PAK4以肺上皮细胞特异性方式缺失的小鼠将受到高氧损伤。终点将包括上皮保护、Akt和不良磷酸化。目的III.探讨KGF抑制跨上皮中性粒细胞迁移的机制及KGF+sRAGE对氧化损伤的保护作用。观察对中性粒细胞浸润、趋化因子分泌、基质金属蛋白酶活性、Syndecan脱落、存活和水肿的影响。目的IV.利用人肺上皮细胞系和原代人肺泡上皮细胞，观察KGF对细胞存活和中性粒细胞迁移的影响。利用人肺上皮细胞系CALU-3和体外形成紧密连接的原代人肺泡上皮细胞，研究KGF的上皮保护作用和抑制跨上皮中性粒细胞迁移的作用。对高氧诱导的细胞死亡和跨上皮中性粒细胞迁移的影响将被跟踪。总而言之，这些研究将解决KGF诱导抗凋亡和抗炎作用的机制。

DESCRIPTION (provided by applicant): Hyperoxic lung injury is associated with widespread epithelial and endothelial injury and cell death together with airway inflammation, edema and hemorrhage. Using an inducible lung-specific transgenic system, we have shown that keratinocyte growth factor (KGF) has a profound protective effect on the lung epithelium and activates the pro-survival Akt pathway both in vivo and in vitro. We have identified intracellular molecules such as PAK4 and p90RSK that interact with the KGF receptor (KGFR) and induce pro-survival mechanisms in lung epithelial cells. We have also found that PAK4 interacts with Raf-1 in mammalian cells. Raf-1 has been recently shown to play a critical role in cell survival. We have identified an additional property of KGF, which is blockade of neutrophil transmigration into the alveolar space, another deleterious event during oxidative injury. In related studies, we have found that the soluble form of the receptor for advanced glycation end products (sRAGE) blocks neutrophil infiltration into the lung during hyperoxic conditions. These observations lead us to hypothesize that KGF and sRAGE inhibit oxidative lung injury through effects on cell survival and anti-inflammatory mechanisms. To test this hypothesis we will: Aim I. Determine the functional importance of the signaling network induced by activated KGFR involving RSK, PAK4 and Raf-1 in epithelial cell responses to hyperoxia. a) Mouse lung epithelial (MLE-12) cells will be subjected to hyperoxia in the presence or absence of KGF and dominant-negative (DN) mutants for KGFR-interacting proteins. Effects on a) subcellular distribution and modification of pro- and anti-apoptotic proteins and on b) Akt and BAD phosphorylation will be followed. Aim II. Investigate the role of KGFR-interacting molecules in Akt and BAD phosphorylation and epithelial protection in vivo, a) Mice with deletions in c-Raf-1 or PAK4 in a lung epithelial cell-specific fashion in the context of inducible KGF transgenic mice will be generated and will be subjected to hyperoxic injury. End-points will include epithelial protection and Akt and BAD phosphorylation. Aim III. Investigate the mechanism by which KGF inhibits transepithelial neutrophil migration and study effects of KGF+sRAGE on protection from oxidative injury. Effects on neutrophil infiltration, chemokine secretion, matrix metalloproteinase activity, syndecan shedding, survival and edema will be followed. Aim IV. Investigate the effect of KGF on cell survival and neutrophil transmigration using a human lung epithelial cell line and primary human alveolar epithelial cells. The epithelial protective effects of KGF and inhibition of transepithelial neutrophil migration will be studied using the human lung epithelial cell line Calu-3 and primary human lung alveolar epithelial cells that can form tight junctions in vitro. Effects on hyperoxia-induced cell death and transepithelial neutrophil migration will be followed. Collectively, these studies will address the mechanisms by which KGF induces anti-apoptotic and anti-inflammatory effects.