CILIARY FUNCTION AND DIFFERENTIATION OF AIRWAY EPITHELIA

描述（由申请人提供） 这个项目的长期目标是提高我们对 粘膜纤毛清除的细胞和分子调节机制， 肺的重要宿主防御机制。 纤毛活动是一个整体 部分粘液纤毛运输和纤毛搏动频率（CBF）的变化是 通常与运输速率的类似变化相关联。 两种广泛 识别细胞信号传导中的第二信使，cAMP和钙（[Ca 2 +]i）， 调节CBF。cAMP已被证明通过cAMP依赖性增加CBF。 激酶介导的事件，可能通过磷酸化纤毛蛋白 命名为P26。 增加[Ca 2 +]i也会刺激CBF，可能是通过 睫状体钙结合蛋白。 因为两种第二信使都增加了CBF， 问题是这些信号通路是否通过 独立的信号转导级联，或者通路是否会聚在 在动力蛋白/微管相互作用之前影响共同靶点。 有证据表明，由于cAMP的升高使CBF 对[Ca 2 +]i变化不敏感。 最简单的解释是 现象是cAMP和Ca 2+信号通路会聚到一个 蛋白质复合物（含有p26），调节CBF。 的磷酸化 该靶标可以改变其Ca 2+亲和力或解离速率。 这项建议旨在 探索p26的身份和功能，并开始检查相互作用 p26和Ca 2+通路之间的关系，通过（1）表征p26并定位它 （2）鉴定外动力蛋白上的钙结合蛋白 （3）评估PKA介导的磷酸化与 p26和CBF增加以及p26磷酸化和Ca 2 +/CBF改变 偶联;以及（4）评估p26与p26之间的因果关系。 磷酸化和CBF变化（部分通过使用转染方法， 将信号蛋白引入纤毛细胞）。 该项目还将 开始应用生理基因组学的方法， 基因表达到生理信号通路。 我们建议使用DNA 微阵列来寻找特定信号通路的成分， 在空气-液体中培养的分化细胞中改变的基因表达 接口，以寻求可靠的，有用的信息，纤毛细胞 基因组水平的生理学。 这些研究的结果将提供 新的和重要的信息的作用，特定的纤毛蛋白， 调节哺乳动物CBF。 此外，使用微阵列的研究将 产生关于特定信号通路的新信息， 识别未知的调节途径的组成部分。

DESCRIPTION (provided by applicant) The long-term goal of this project is to improve our understanding of the cellular and molecular regulation mechanisms of mucociliary clearance, an important host defense mechanism of the lung. Ciliary activity is an integral part of mucociliary transport and changes in ciliary beat frequency (CBF) are often associated with similar changes in transport rates. Two widely recognized second messengers in cell signaling, cAMP and calcium ([Ca2+]i), regulate CBF. cAMP has been shown to increase CBF through a cAMP-dependent kinase- mediated event, possibly by phosphorylating a ciliary protein designated p26. Increasing [Ca2+]i also stimulates CBF, likely through a ciliary Ca2+-binding protein. Because both second messengers increase CBF, the question arises whether these signaling pathways regulate CBF through independent signal transduction cascades or whether the pathways converge at some level to affect a common target prior to dynein/microtubule interaction. Evidence suggests that they converge because elevations in cAMP render CBF less sensitive to [Ca2+]i changes. The simplest hypothesis to explain this phenomenon is that the cAMP and Ca2+ signaling pathways converge onto a protein complex (containing p26), which regulates CBF. The phosphorylation of this target may change its Ca2+ -affinity or off-rate. This proposal seeks to explore the identity and function of p26 and begin to examine interactions between p26 and the Ca2+ pathway by (1) characterizing p26 and localizing it within the cilium; (2) identifying a calcium-binding protein on outer dynein arms; (3) evaluating the correlation between PKA-mediated phosphorylation of p26 and increases in CBF as well as p26 phosphorylation and altered Ca2+/CBF coupling; and (4) evaluating a cause-effect relationship between p26 phosphorylation and CBF changes (partially by using transfection methods to introduce signaling proteins into ciliated cells). The project will also begin to apply the approach of physiological genomics, which tries to link gene expression to physiological signaling pathways. We propose to use DNA microarrays to search for components of specific signaling pathways through altered gene expression in differentiating cells cultured at the air-liquid interface in order to seek reliable, useful information about ciliated cell physiology at the genomic level. The results of these studies will provide new and important information on the role of specific ciliary proteins in regulating mammalian CBF. Furthermore, the studies using microarrays will generate new information on specific signaling pathways and may well serve to identify components of regulatory pathways that were unknown.