Mucin hyperproduction is a common component of obstructive airways disease and comprises a principle problem faced by patients and their attending physicians. A major long-term goal of this laboratory is the identification of molecular targets for pharmacologic therapies against mucin hypersecretion. Using SPOC1 cells as a model for airway mucin secretion, we have shown that phospholipase C-coupled P2Y2 receptors localized in the apical membrane form the dominate regulatory pathway in these cells. Because all airway epithelial cells likely possess P2Y2 receptors for extracellular ATP and UTP, targets for the selective inhibition of mucin secretion need to be located at points distal to activation of PLC, and IP3 and DAG generation. The two downstream elements of the PLC pathway, Ca2+-protein kinase C (PKC), appear to be independent in their activation of mucin release, and preliminary data suggest strongly that nPKCdelta, a Ca2+-insensitive isoform, is uniquely activated by agonist. This proposal will test whether the Ca2+ and PKC pathways leading to mucin secretion are fully additive and independent, or whether they converge at a rate limiting step proximal to mucin granule/plasma membrane fusion and exocytosis. A likely candidate for this proximal barrier to secretion is the cortical cytoskeleton, comprised of actin microfilaments.
Specific Aim 1 will use wild-type and mutant nPKCdelta retroviral expression vectors to test whether nPKCdelta, in fact, mediates the effects of agonist of mucin secretion, and it will examine the means by which nPKCdelta is activated.
Specific Aim II tests the independence between Ca2+- and PKC-activated mucin secretory pathways by probing the transit of mucin granules across the cortical microfilament barrier and the plasma membrane. We focus on the roles of scinderin, a gelsolin-related, F-acting severing enzyme, and Rab3 isoforms, respectively. In each case, we will test whether these elements participate in the regulation of agonist-stimulated exocytosis and whether they lie in the pathways modulated by nPKCdelta and/or Ca2+. Lastly, we will exploit the different binding kinetics of BAPTA and EGTRA to test the degree of independence between PKC and Ca2+ in the final steps of exocytosis.
| Kreda, Silvia M; Davis, C William; Rose, Mary Callaghan (2012) CFTR, mucins, and mucus obstruction in cystic fibrosis. Cold Spring Harb Perspect Med 2:a009589 |
| Abdullah, Lubna H; Wolber, Cédric; Kesimer, Mehmet et al. (2012) Studying mucin secretion from human bronchial epithelial cell primary cultures. Methods Mol Biol 842:259-77 |
| Schmid, Andreas; Clunes, Lucy A; Salathe, Mathias et al. (2011) Nucleotide-mediated airway clearance. Subcell Biochem 55:95-138 |
| Tuvim, Michael J; Mospan, Andrea Rossi; Burns, Kimberlie A et al. (2009) Synaptotagmin 2 couples mucin granule exocytosis to Ca2+ signaling from endoplasmic reticulum. J Biol Chem 284:9781-7 |
| Davis, C William; Lazarowski, Eduardo (2008) Coupling of airway ciliary activity and mucin secretion to mechanical stresses by purinergic signaling. Respir Physiol Neurobiol 163:208-13 |
| Zhu, Yunxiang; Ehre, Camille; Abdullah, Lubna H et al. (2008) Munc13-2-/- baseline secretion defect reveals source of oligomeric mucins in mouse airways. J Physiol 586:1977-92 |
| Davis, C William; Dickey, Burton F (2008) Regulated airway goblet cell mucin secretion. Annu Rev Physiol 70:487-512 |
| Rossi, Andrea H; Salmon, Wendy C; Chua, Michael et al. (2007) Calcium signaling in human airway goblet cells following purinergic activation. Am J Physiol Lung Cell Mol Physiol 292:L92-8 |
| Ehre, Camille; Zhu, Yunxiang; Abdullah, Lubna H et al. (2007) nPKCepsilon, a P2Y2-R downstream effector in regulated mucin secretion from airway goblet cells. Am J Physiol Cell Physiol 293:C1445-54 |
| Abdullah, Lubna H; Davis, C William (2007) Regulation of airway goblet cell mucin secretion by tyrosine phosphorylation signaling pathways. Am J Physiol Lung Cell Mol Physiol 293:L591-9 |
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