KGF and Protection from Hyperoxic Lung Injury

描述(申请人摘要)：高氧性肺损伤与 肺泡上皮广泛解体。无数的研究 角质形成细胞生长因子(KGF)的保护作用 氧化剂引起的肺损伤。我们发现KGF能诱导细胞外信号转导通路激活 肺上皮细胞丝氨酸-苏氨酸激酶Akt与PI3-激酶的关系 并导致不良的磷酸化。使用酵母双杂交系统，我们有 其产物与猪瘟病毒胞浆结构域相互作用的分离的cDNA克隆 KGFR。其中两个克隆编码p90RSK和PAK4，有4个 新的c DNA克隆。我们的观察使我们假设：1)。KGF 通过肺保护作用预防高氧性肺损伤 上皮细胞。2)。AKT激活在KGF的保护作用中起重要作用。 3)。KGF诱导的信号转导涉及特异性的相互作用 含有KGFR胞浆结构域的胞内蛋白。向我们的 假设我们将：目标I.研究免疫保护机制 利用过量表达KGF或KGF的可诱导转基因小鼠诱导氧化损伤 AKT的一种构成上活跃的形式。1)KGF的表达将在 高氧暴露前和暴露期间的不同时间。转基因动物的肺 将对小鼠进行以下分析：a)组织学和电子显微镜，b)生化 检测(蛋白质泄漏、脂质过氧化、氧化还原状态)，c)基因表达 研究(表面活性物质、细胞死亡相关基因)，d)肺研究 炎症(细胞因子/趋化因子表达)2)表达 以诱导方式构成的AKT的活性形式将类似地 分析过了。目的II.与之相互作用的细胞内蛋白的特性 KGFR。1)在多个独立分离物中鉴定出一个新的cdna克隆。 并且在肺和上皮细胞中高表达将是其特征。 2)a)哺乳动物细胞中的相互作用将被证实为 免疫共沉淀实验，以及b)相互作用是否需要 KGFR和c)的酪氨酸激酶活性将具有生物学意义 由突变体在细胞培养和KGF小鼠中的表达决定 腺病毒基因转移技术。目标三.结构与功能的关系 KGFR介导的高氧性肺损伤保护作用的研究。1)特定酪氨酸 Trk-kgfr嵌合构建体的细胞质区域中的残基将是 多角化以确定KGFR中的结构-功能关系 大鼠II型细胞和小气道上皮细胞及多种分子 以及生化方法。2)过表达KGF的小鼠将被感染 编码特定KGFR突变体的重组腺病毒检测其 对体内高氧损伤的影响。

DESCRIPTION (Applicant's abstract): Hyperoxic lung injury is associated with extensive disintegration of the alveolar epithelium. Numerous studies demonstrate a protective role of keratinocyte growth factor (KGF) in oxidant-induced lung injury. We have found that KGF induces the activation of the serine-threonine kinase Akt in lung epithelial which involves PI 3-kinase and results in BAD phosphorylation. Using the yeast two-hybrid system, we have isolated cDNA clones whose products interact with the cytoplasmic domain of KGFR. Two of these clones encode the kinases p90RSK and PAK4 and there are 4 novel cDNA clones. Our observation leads us to hypothesize that: 1). KGF prevents hyperoxic lung injury through its protective effects on lung epithelial cells. 2). Akt activation plays a major role in protection by KGF. 3). KGF-induced signal transduction involves interaction of specific intracellular proteins with the cytoplasmic domain of KGFR. To address our hypothesis we will: Aim I. Investigate the mechanisms of protection from oxidant-induced injury by using inducible transgenic mice overexpressing KGF or a constitutively active form of Akt. 1) KGF expression will be induced at different times before and during hyperoxic exposure. The lungs of transgenic mice will be analyzed by: a) histology and electron microscopy, b) biochemical assays (protein leak, lipid peroxidation, redox status), c) gene expression studies (surfactant, cell death-associated genes), d) studies of lung inflammation (cytokine/chemokine expression) 2) Transgenic mice expressing a constitutively active form of Akt in an inducible fashion will be similarly analyzed. Aim II. Characterization of intracellular proteins that interact with the KGFR. 1) One novel cDNA clone identified in multiple independent isolates and highly expressed in the lung and in epithelial cells will be characterized. 2) a) Interactions in mammalian cells will be confirmed by co-immunoprecipitation experiments, and whether the interactions b) require the tyrosine kinase activity of KGFR and c) have biologic significance will be determined by expressions of mutants in cell culture and in KGF mice using adenoviral gene transfer techniques. Aim III. Structure-function relationships of KGFR-mediated protection from hyperoxic lung injury. 1) Specific tyrosine residues in the cytoplasmic domain of a trk-KGFR chimeric construct will be mutangenized to determine the structure-function relationships in KGFR using rat type II cells and small airways epithelial cells and a variety of molecular and biochemical approaches. 2) KGF overexpressing mice will be infected with recombinant adenovirus encoding particular KGFR mutants to determine their effects on hyperoxic injury in vivo.