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.
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