KGF and Protection from Hyperoxic Lung Injury

描述（由申请人提供）：高氧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、水肿和出血相关。使用可诱导的肺特异性转基因系统，我们已经表明，角质细胞生长因子（KGF）对肺上皮细胞具有深远的保护作用，并在体内和体外激活促生存Akt通路。我们已经确定了细胞内分子，如PAK4和p90RSK与KGF受体（KGFR）相互作用，并诱导肺上皮细胞中的促生存机制。我们还发现PAK4在哺乳动物细胞中与Raf-1相互作用。Raf-1最近被证明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，即阻断中性粒细胞迁移到肺泡腔，这是氧化损伤期间的另一个有害事件。在相关的研究中，我们发现晚期糖基化终产物（sNADP）受体的可溶性形式在高氧条件下阻断中性粒细胞浸润到肺中。这些观察结果使我们假设KGF和sodium通过影响细胞存活和抗炎机制来抑制氧化性肺损伤。为了验证这一假设，我们将：目的一。确定在上皮细胞对高氧的反应中由涉及RSK、PAK 4和Raf-1的活化KGFR诱导的信号网络的功能重要性。a）小鼠肺上皮（MLE-12）细胞在存在或不存在KGF和KGFR相互作用蛋白的显性阴性（DN）突变体的情况下经受高氧。将跟踪对a）促凋亡蛋白和抗凋亡蛋白的亚细胞分布和修饰以及对B）Akt和BAD磷酸化的影响。Aim II.研究KGFR相互作用分子在体内Akt和BAD磷酸化和上皮保护中的作用。a）在诱导型KGF转基因小鼠的背景下，将产生以肺上皮细胞特异性方式缺失c-Raf-1或PAK 4的小鼠，并使其经受高氧损伤。终点将包括上皮保护和Akt和BAD磷酸化。Aim III.研究KGF抑制中性粒细胞跨上皮迁移的机制，并研究KGF + sodium对氧化损伤的保护作用。将跟踪对中性粒细胞浸润、趋化因子分泌、基质金属蛋白酶活性、多配体蛋白聚糖脱落、存活和水肿的影响。目标四。使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。将使用人肺上皮细胞系Calu-3和可在体外形成紧密连接的原代人肺泡上皮细胞研究KGF的上皮保护作用和对跨上皮中性粒细胞迁移的抑制。将跟踪对高氧诱导的细胞死亡和跨上皮中性粒细胞迁移的影响。总的来说，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.