Excess mucus secretion in airway is associated with asthma and airway inflammation, obstructive respiratory diseases, and cystic fibrosis. Cellular mechanism controlling hypersecretion of mucin (the glycoprotein component of mucus) by airway epithelial cells have not been elucidated. In this application, we propose that MARCKS (MYRISTOYLATED ALANINE-RICH C KINASE SUBSTRATE) PROTEIN is a key intracellular signaling molecule playing a central role controlling mucin hypersecretion. It is proposed that MARCKS is the intracellular link between secretagogue stimulation at the cell surface and release of mucin granules by airway secretory cells. The specific hypothesis to be addressed is that mucin secretagogues activate two separate protein kinases in goblet cells: PKC (protein kinase C) and PKG (cGMP-dependent protein kinase). PKC phosphorylates MARCKS, causing its release from the inner face of the plasmalemma to the cytoplasm, where it is targeted to and binds to mucin granule membranes. PKG activates a protein phosphatase which dephosphorylates MARCKS, stabilizing its attachment to the granules membrane and allowing it to bind actin filaments, contraction of which moves the granules to the cell surface for exocytotic release. The experiment in this proposal will determine if the above MARCKS-dependent mechanism modulates stimulated mucin secretion in NORMAL HUMAN BRONCHIAL EPITHELIAL (NHBE) CELLS maintained in air/liquid interface primary culture, a system developed in this laboratory to maintain human airway epithelial cells in vitro with differentiated structure and function. The experiment will examine critical steps in this hypothetical pathway as to their role in stimulated mucin secretion: The requirement for PKC and PKG activation (AIM 1); a direct, central role for MARCKS in the secretory pathway (AIM 2); and the intracellular mechanism of MARCKS action: phosphorylation, translocation, targeting to intracellular mucin granules and dephosphorylation of MARCKS (AIM 3). These studies will demonstrate the existence of the heretofore undescribed major intracellular pathway modulating a primary respiratory defense mechanism, mucin secretion.
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