KGF and Protection from Hyperoxic Lung Injury

描述（由申请人提供）：高氧性肺损伤与广泛的上皮和内皮损伤和细胞死亡以及气道炎症、水肿和出血有关。利用可诱导的肺特异性转基因系统，我们在体内和体外均证明了角质细胞生长因子（KGF）对肺上皮具有深远的保护作用，并激活促存活Akt通路。我们已经鉴定出细胞内分子，如PAK4和p90RSK，它们与KGF受体（KGFR）相互作用，并诱导肺上皮细胞的促存活机制。我们还发现PAK4在哺乳动物细胞中与Raf-1相互作用。最近发现Raf-1在细胞存活中起关键作用。我们已经确定了KGF的另一个特性，即阻断中性粒细胞向肺泡间隙的迁移，这是氧化损伤期间的另一个有害事件。在相关研究中，我们发现晚期糖基化终产物受体（sRAGE）的可溶性形式在高氧条件下阻断中性粒细胞向肺的浸润。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抑制氧化性肺损伤。为了验证这一假设，我们将：目的1 .确定激活的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，包括RSK、PAK4和Raf-1。a)小鼠肺上皮细胞（MLE-12）在存在或不存在KGF和kgfr相互作用蛋白的显性阴性（DN）突变体的情况下都会遭受高氧。对a)亚细胞分布和促凋亡和抗凋亡蛋白修饰的影响，以及对b) Akt和BAD磷酸化的影响。目的二世。研究kgfr相互作用分子在体内Akt和BAD磷酸化和上皮细胞保护中的作用，a)在诱导的KGF转基因小鼠环境下，会产生肺上皮细胞特异性缺失c-Raf-1或PAK4的小鼠，并将遭受高氧损伤。终点包括上皮保护和Akt和BAD磷酸化。第三目标。探讨KGF抑制经上皮中性粒细胞迁移的机制，研究KGF+sRAGE对氧化损伤的保护作用。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。目的四：利用人肺上皮细胞系和原代人肺泡上皮细胞，研究KGF对细胞存活和中性粒细胞迁移的影响。我们将利用人肺上皮细胞系Calu-3和能在体外形成紧密连接的人肺泡上皮细胞，研究KGF对上皮的保护作用和对经上皮中性粒细胞迁移的抑制作用。对高氧诱导的细胞死亡和经上皮中性粒细胞迁移的影响将被跟踪。总的来说，这些研究将探讨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.