DESIGN AND TRANSGENIC ANALYSIS OF CELLULAR INHIBITORS

我们项目的中心假设是，短肽可以用作有效的，特定的抑制剂，可以针对期望的细胞器和细胞类型，以精确地阐明细胞途径。我们已经证明，II型细胞中钙调蛋白的核功能对小鼠肺发育至关重要。高度扁平的I型细胞为O2/CO2交换提供了广阔的细胞表面。肺泡对维持氧稳态至关重要。I型细胞对来自环境挑战的损伤极其敏感，它们不分裂。肺损伤后，II型细胞最终分化，以新的I型细胞重新填充肺泡上皮。本项目的研究将使用新的转基因方法在体内研究CaM激酶II和caln调节通路在肺发育和II型细胞对肺损伤的反应中的作用。CaM kinase II和CalN是介导Ca/2+/CaM调控细胞分泌、离子通量、DNA复制和RNA转录等细胞活动的主要酶系统。在转基因小鼠中进行了研究，其中SP-C（表面活性剂蛋白C）启动子用于指导合成的显性负基因的表达，这些基因可以中和肺II型上皮细胞中CaM激酶II和CalN的功能。这些合成基因编码肽串联体，这些肽串联体结合到催化亚基的活性位点上，并以细胞特异性的方式抑制其靶酶。这些抑制肽串联体靶向肺上皮将导致产生独特的表型，其中Ca/2+激活的信号转导系统被改变。本项目将评估CaM激酶II和CaIN在肺发育和氧化应激、臭氧和二氧化硅诱导损伤的生理反应中的作用。CaM激酶II和CalN可能是正常肺功能如液体和表面活性剂分泌所必需的。预计将开发出能够改变肺顺应性、粘液充血和肺纤维化的独特小鼠系。这些病理生理状况会导致肺泡气体交换减少，从而导致低氧血症、血浆pH失衡和身体残疾。

The central hypothesis of our project has been that short peptides can be used as potent, specific inhibitors which can be targeted to desire organelles and cell types in order to precisely elucidate cellular pathways. We have shown that the nuclear function of calmodulin in type II cells is critical for murine lung development. The highly flattened type I cells provide a vast cellular surface for O2/CO2 exchange. Lung alveoli are critical for the maintenance of oxygen homeostasis. Type I cells are extremely sensitive to injury from environmental challenges and they do not divide. Following lung damage, type II cells terminally differentiate to repopulate the alveolar epithelium with new type I cells. The research in this project will use novel transgenic approaches to study, in vivo, the role of CaM kinase II- and CalN-regulated pathways involved in lung development and type II cell response to lung injury. CaM kinase II and CalN are the major enzyme systems which mediate Ca/2+/CaM regulation of cellular activities such as secretion, ion fluxes, DNA replication and RNA transcription. Studies are performed in transgenic mice in which the SP-C (surfactant protein C) promoter is used to direct expression of synthetic dominant-negative genes which neutralize the function of CaM Kinase II and CalN in lung type II epithelial cells. These synthetic genes encode peptide concatemers which bind to the active site in the catalytic subunit and cause inhibition of their targeted enzymes in a cell-specific manner. The targeting of these inhibitory peptide concatemers to the lung epithelium will lead to the generation of unique phenotypes in which Ca/2+- activated signal transduction systems are altered. This project will evaluate the role of CaM kinase II and CaIN in lung development and physiological responses to oxidative stress, ozone and silica-induced injury. CaM kinase II and CalN may be required for proper lung function such as fluid and surfactant secretion. It is anticipated that unique mouse lines will be developed that have altered lung compliance, mucous congestion and lung fibrosis. These pathophysiological conditions will result in decreased gas exchange in the alveolus which will cause hypoxemia, plasma pH imbalance and physical disability.