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 粘液纤毛清除的细胞和分子调控机制 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 通常与运输率的类似变化有关。两个广泛的 识别细胞信号中的第二信使，cAMP和钙([Ca2+]i)， 调节CBF。CAMP已被证明通过cAMP依赖增加CBF 激酶介导的事件，可能是通过使纤毛蛋白磷酸化 指定为第26页。增加[Ca~(2+)]i也刺激脑血流，可能是通过 纤毛钙结合蛋白。因为两个第二信使都增加了CBF， 问题是，这些信号通路是否通过 独立的信号转导级联或通路是否汇聚在 在动力蛋白/微管相互作用之前影响共同靶点的某种程度。 有证据表明，它们之所以会聚是因为营地的海拔高度导致了CBF 对[Ca~(2+)]i变化不太敏感。解释这一点的最简单的假设是 现象是cAMP和钙信号通路汇聚成一个 蛋白质复合体(包含P26)，调节CBF。蛋白的磷酸化 这个靶点可能会改变它的钙亲和力或去除率。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和钙途径之间的关系：(1)P26的特性和定位 纤毛内；(2)鉴定外动力蛋白上的钙结合蛋白 ARMS；(3)评估PKA介导的磷酸化之间的相关性。 P26和CBF增加以及P26磷酸化和改变钙/CBF 以及(4)评价P26之间的因果关系 磷酸化和CBF的变化(部分是通过使用转染法 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，试图将 基因表达与生理信号通路的关系。我们建议使用DNA 通过微阵列搜索特定信号通路的组件 气液混合培养中分化细胞基因表达的变化 接口，以便寻找有关纤毛细胞的可靠、有用的信息 基因组水平的生理学。这些研究的结果将提供 关于特定纤毛蛋白在眼球运动中作用的新的重要信息 调节哺乳动物的脑血流量。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，并可能很好地服务于 确定未知的调控途径的组成部分。

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.