Regulation of Mucociliary Clearance in Airways
Figure 1. Effectors in regulated mucin granule exocytosis from goblet cells.
Efficient removal of inhaled particulates and pathogens from the lungs by the mucociliary clearance (MCC) system depends on the coordination of three cellular activities in the airways epithelium: [i] mucins are secreted to form mucus in which particulates are entrapped; [ii] coordinated ciliary activity propels the mucus-ensnared material toward the mouth; and [iii] transepithelial ion and fluid secretion and absorption mechanisms control the availability of water for mucin hydration, as well as the thickness of the periciliary fluid layer bathing the luminal surface of the airways necessary for efficient ciliary beating. The Davis Lab focuses on the role of mucus and mucins in MCC, especially the regulation of mucin secretion at the cell and molecular levels (Fig. 1).
Mucins are polymeric glycoproteins, with monomeric molecular weights of ~10 MDa and polymeric molecular weights of several 10s of MDa, that are released by exocytosis from goblet cells and submucosal glands into the airway lumen. The glands are controlled by the nervous system through acetylcholine and VIP neurons, whereas the goblet cells, on which we focus, are regulated by local signaling within the luminal compartment. We have used a variety of cell culture and mouse models to show that mucin secretion in human, rat, mouse, and canine airways is under the powerful control of purinergic agonists (ATP, UTP) which interact from the lumen with apical membrane P2Y2 purinoceptors. In all the models, Ca2+ and PKC activity are involved in the regulation of Ca2+ secretion, consistent with the linkage of the P2Y2-R with phospholipase C. We have recently shown that Ca2+ and PKC act together, with the gelsolin-related actin filament severing enzyme, scinderin, and MARCKS, respectively, to regulate disassembly of the actin cytoskeleton and initiate the mucin granule exocytic process. Click on the following links to access recent reviews, one written with UNC CF Center colleague, Eduardo Lazarowski, the other with Burton Dickey a collaborator at MD Anderson Cancer Center in Houston. The first summarizes our current understanding of the regulation of MCC by mechanical stresses via purinergic agonists [Davis & Lazarowski]; the second is a comprehensive review covering regulated mucin granule exocytosis from goblet cells, progressing from receptor activation to assembly and activation of the SNARE complex [Davis & Dickey].
Figure 2. (A,B) KO of Munc13-2 from mouse airways causes accumulation of mucins in Clara cells. (C) Although Munc13-2 is imp. in exocytic priming, the cells retain secretory response, indicating priming by other priming protein expressed, Munc13-4.
Regulated Mucin Secretion
Presently, the Davis Lab is studying the roles of accessory proteins that regulate the SNARE complex following cell activation -- these are the events that comprise the last steps of regulated secretion. The background for this work was provided by findings from the Davis and Dickey Labs with mice deficient for Munc13-2 (Zhu, et al, 2008) and Synaptotagmin-2 (Syt2; Tuvim, et al., 2009), respectively, experimental models that offered a profound view into regulated mucin secretion. In the mouse lung, in contrast to the human, the primary secretory cell is the Clara cell which under control conditions secretes a lipoprotein of uncertain function. Under inflammatory conditions, though, Clara cells transform metaplastically into goblet cells, which resemble closely those in human airway. Control mice therefore appear to be devoid of PAS (periodic acid-Schiff’s) staining (Fig 2A, top panel), whereas mice with mucous metaplasia, induced by any of the many different inflammatory pathways, are PAS+. Notably, the airways of the Munc13-2 null mouse are PAS+ (Fig 2b, bottom panel); however, the presence of significant quantities of mucin signifies, not mucous metaplasia, but a regulated exocytic pathway, dependent on Munc13-2, that appears to operate at baseline in parallel with the agonist-stimulated pathway(s). Following up on the notion that mucins are secreted at baseline in WT mice, we used reagents more sensitive than PAS to show that, in fact, Clara cells secreted mucins under control conditions. As Fig 2 shows, Munc13-2 is not essential for agonist-stimulated secretion, and exocytosis in this case appears to be supported by Munc13-4, the other exocytic priming protein expressed in goblet cells. In contrast, in the Syt2 null mouse agonist-stimulated mucin secretion was abolished; however, there was no mucin accumulation at baseline, indicating that Syt2 is not essential for the basal secretory pathway. These two papers have given rise to the notion that there are two, or more, pathways for the release of mucins onto airway surfaces. Minimally, one subserves secretion at baseline, likely in the absence of agonist; the other, regulates secretion in response to agonist (ATP or UTP).
These pathways, which seem to operate in parallel, may share the same SNARE complex (syntaxin, vamp, snap25), but different regulatory accessory proteins regulate their respective activities. Munc13-2 (and, redundantly, Munc13-4) and an unidentified Ca2+ sensor control the basal exocytic pathway, whereas the agonist pathway is regulated by Munc13-4 (and, redundantly, Munc13-2) and Syt2. In current work, with the support of an NIH R01 grant, the Davis and Dickey Labs are testing this hypothesis.
Post-secretory Maturation of Mucins
Existing and emerging evidence from several sources suggest that mucins likely are packaged in a highly ordered fashion within secretory granules through extensive disulfide crosslinks between N-terminal D domains within individual polymer molecules. The North American Cystic Fibrosis Foundation has funded a Mucus Clearance Consortium whose charge, in part, is to test this hypothesis and to determine whether CF pathogenesis, in part, results from a mucin that is not processed properly after it is secreted. The Davis and Kesimer/Sheehan Labs are collaborating with other investigators at other institutions to test this hypothesis.
The Davis Lab also has substantial collaborative relationships with others in the CF Center in a variety of areas, but mostly relating to the inter-relationships between mechanical stresses, ATP secretion, intracellular Ca2+ and cAMP in the regulation of ciliary activity and mucin secretion, and the physical relationships between the mucus, water, and the luminal surface of the airways during mucociliary clearance.
C William Davis, PhD, Professor of Cell & Molecular Physiology and Medicine
Bill Davis holds a primary appointment in the Department of Cell & Molecular Physiology, and a joint appointment in Pulmonary Medicine. His laboratory is located within the Cystic Fibrosis/Pulmonary Research & Treatment Center.
1969 BA, San Francisco State University
1973 MA, San Francisco State University
1978 PhD, U. of North Carolina at Chapel Hill
1983 Post Doctorate, U. of North Carolina at Chapel Hill
Published Book Chapters and Reviews (Selected & Recent; of 12 Total Chapters and Reviews)
1. Davis, C.W. 1997. Goblet cells: physiology and pharmacology. In: Airway Mucus: Basic Mechanisms and Clinical Perspectives. Eds. D.F. Rogers and M.I. Lethem. Birhäuser Verlag, Basel, pp. 149-177.
2. Davis, C.W. and S.H. Randell. 2001. Airway goblet and mucous cells: identical, similar, or different?. In: Cilia and Mucus: From development to respiratory disease. Ed. M. Salathe. Marcel Dekker, N.Y., p.195-210.
3. Davis, C.W. 2002. Regulation of mucin secretion from in vitro cellular models. Novartis Found Symp. 248:113-25; discussion 125-31, 277-82.
4. Davis, C.W. and B.F. Dickey. 2008. Regulated airway goblet cell mucin secretion. Ann. Rev. Physiol. 70:487-512.
5. Davis, C.W. and E.R. Lazarowski. 2008. Coupling of airway ciliary activity and mucin secretion to mechanical stresses by purinergic signaling. Resp. Physiol. Neurobiol. ePub May 28, PMID 18635403
Cystic Fibrosis Foundation Therapeutics, Inc., Cystic Fibrosis Foundation, National Institutes of Health (NIH), Astra/Zeneca.
Please see Pubmed feed in the righthand column for links to current publications.
Dr. C William Davis, Professor of Cell & Molecular Physiology of Medicine
Dr. Lubna Abdullah, Research Associate
Laura Moussa (Left) and Summer Bailey (Right), Research Technicians
The University of North Carolina at Chapel Hill
Chapel Hill, NC 27599
Campus Box #7248
Phone: (919) 966-7060
Fax: (919) 966-5178