The secreted gel-forming mucins of the airway provide important protective functions through the clearance of inhaled pathogens and particulates;however, mucin hypersecretion causes airflow obstruction and airway injury in most diseases of the airways. Therefore, tight control of mucin production and secretion is critical for airway homeostasis. Airway goblet cell mucin secretion is principally controlled by signaling pathways downstream of P2Y2 purinoceptors, though additional G-protein coupled receptors and receptor tyrosine kinases may also play significant roles. Activation of P2Y2 receptors by nucleotides in the airway liquid layer leads to generation of the second messengers diacylglyerol (DAG) and calcium (Ca2+) that together result in robust mucin secretion. Significantly, the majority of mucins secreted in a lung are released, continuously, at baseline, by pathways whose regulation is unknown. Components of the cellular exocytic machinery known to respond to agonist-generated second messengers are the Munc13 priming proteins that bind DAG and Ca2+, and the SNARE-triggering Synaptotagmin (Syt) proteins that bind phospholipids and Ca2+. We have found that the absence of one of the two Munc13 proteins expressed in the airways, Munc13-2, results in partial defects in baseline and stimulated mucin secretion in mice: this mouse model therefore offers the first experimental tool useful in unraveling regulated baseline mucin secretion. Munc13-4, the other isoform expressed, presumably regulates agonist stimulated mucin secretion;however, our preliminary data indicate it may play no role in baseline secretion. In contrast, the absence of Syt-2 results in a profound defect in stimulated secretion, but we deduce a paradoxical increase in baseline mucin secretion. Additional Syt isoforms are expressed in mouse and human mucous cells whose interactions with Syt-2 might explain these paradoxical results. We hypothesize that baseline and agonist stimulated mucin secretion is controlled by signaling pathways upstream of Munc13 and Syt proteins in airway mucin secreting cells, and that specific Munc13/Syt pairs partition baseline and agonist stimulated mucin secretions. Mouse models will be used to test this hypothesis and to determine the precise mechanisms of regulation of airway mucin secretion.
In Specific Aim 1, we will determine the roles of Munc13-2 and Munc13-4 in airway mucin secretion in vivo and in vitro, and identify how they are regulated and how they contribute individually to baseline and agonist stimulated pathways.
For Specific Aim 2, we will determine the roles of Syt-2 and Syt-7 in airway mucin secretion in vivo and in vitro, identify whether and how they are regulated to contribute differentially to baseline and agonist stimulated pathways, and determine whether there are significant functional interactions between the Syt and Munc13 isoforms of interest.

Public Health Relevance

Mucin hypersecretion, resulting in a overproduction of mucus in the airways of the lungs, substantially increases patient morbidity and mortality in literally all the airways diseases, common ones (chronic bronchitis and asthma) and orphan ones (cystic fibrosis and primary ciliary dyskinesia) alike, irrespective of the initiating environmental and/or genetic insult. Consequently, it is important to understand the molecular pathways by which mucins are secreted and by which their secretion is regulated, to enable effective therapeutic design and treatment. This project will use gene knockout and overexpression mouse models to reveal the roles of two kinds of proteins involved in regulating mucin secretion, Munc13, which is responsible for priming the secretory apparatus, and synaptotagmin, which acts as the final intracellular calcium sensor and trigger of secretion.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL097000-04
Application #
8435548
Study Section
Lung Cellular, Molecular, and Immunobiology Study Section (LCMI)
Program Officer
Banks-Schlegel, Susan P
Project Start
2010-04-01
Project End
2014-02-28
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
4
Fiscal Year
2013
Total Cost
$468,790
Indirect Cost
$78,750
Name
University of North Carolina Chapel Hill
Department
Physiology
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Rodarte, Elsa M; Ramos, Marco A; Davalos, Alfredo J et al. (2018) Munc13 proteins control regulated exocytosis in mast cells. J Biol Chem 293:345-358
Nguyen, Long P; Al-Sawalha, Nour A; Parra, Sergio et al. (2017) ?2-Adrenoceptor signaling in airway epithelial cells promotes eosinophilic inflammation, mucous metaplasia, and airway contractility. Proc Natl Acad Sci U S A 114:E9163-E9171
Zhu, Yunxiang; Abdullah, Lubna H; Doyle, Sean P et al. (2015) Baseline Goblet Cell Mucin Secretion in the Airways Exceeds Stimulated Secretion over Extended Time Periods, and Is Sensitive to Shear Stress and Intracellular Mucin Stores. PLoS One 10:e0127267
Ren, Binhui; Azzegagh, Zoulikha; Jaramillo, Ana M et al. (2015) SNAP23 is selectively expressed in airway secretory cells and mediates baseline and stimulated mucin secretion. Biosci Rep 35:
Evans, Christopher M; Raclawska, Dorota S; Ttofali, Fani et al. (2015) The polymeric mucin Muc5ac is required for allergic airway hyperreactivity. Nat Commun 6:6281
Roy, Michelle G; Livraghi-Butrico, Alessandra; Fletcher, Ashley A et al. (2014) Muc5b is required for airway defence. Nature 505:412-6
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
Piccotti, Lucia; Dickey, Burton F; Evans, Christopher M (2012) Assessment of intracellular mucin content in vivo. Methods Mol Biol 842:279-95
Dickey, B F (2012) Biochemistry. Walking on solid ground. Science 337:924-5
Kim, Kyubo; Petrova, Youlia M; Scott, Brenton L et al. (2012) Munc18b is an essential gene in mice whose expression is limiting for secretion by airway epithelial and mast cells. Biochem J 446:383-94