Efficient removal of inhaled particulates and pathogens from the lungs by the mucociliary clearance system depends on the coordination of three cellular activities: [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.  We have studied selected aspects of this system, with a major focus directed at understanding the regulation of mucin secretion at the cell and molecular levels (Fig. 1).

Fig 2.  Effects of nPKCd (top) or nPKCe (bottom) knockout on agonist-induced mucin secretion from perfused mouse tracheas (Ehre, et al.,2007).

Mucin is a very high molecular weight glycoconjugate that is released by exocytosis into the airway lumen. We have used a variety of cell culture and mouse models to show that mucin secretion in human, rat, 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 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 preparatory to mucin granule exocytosis.

PKC is a diverse family of enzymes with 3 subfamilies which differ primarily in the mode of activation:  conventional isoforms are activated by diacylglycerol (DAG; or its mimic, PMA) and  Ca²⁺ by virtue of C1 and C2 domains, respectively;  novel isoforms possess C2 domains that are Ca²⁺-insensitive, so are activated soleley by DAG;  atypical isoforms, whose C2 domains are absenst and whose C1 domains are uniquely DAG-insensitive, are activivated by phosphorylation and do not participate directly in receptor-mediated cellular responses.  In a long series of experiments we have shown that of the 5 PKC isoforms expressed in goblet cells (cPKCα, nPKCδ, nPKCε, nPKCη, aPKCζ), only nPKCε appears to mediate agonist-stimulated mucin secretory responses.  Interestingly, nPKCδ is activated by purinergic agonists, and it translocates from the cytosol to the membrane fraction;  however, neither overexpression of nPKCδ in SPOC1 cells, nor knockout of nPKCδ in mice has any measurable effect on stimulated mucin secretion.  In contrast, overexpression of nPKCε increases mucin secretion from SPOC1 cells, and its knockout in mice nearly abolishes secretion from tracheas (Fig 2).

Presently, we are studying the role of the exocytic priming protein, MUNC13-2, in mucin secretion.  This study has already yielded the surprising notion that Munc13-2 regulates a baseline secretory pathway (Fig 3), and it has identified the Clara cell in the mouse airway as the source of mucin under control conditions.

Fig 3 (A,B):  KO of Munc13-2 from mouse airways causes the accumulation of mucins in Clara cells (AB-PAS stain).  Note the relative lack of staining in the control airway.   (C):  Even though Munc13-2 is important in exocytic priming, the cells retain a secretory response, likely indicating priming by the other priming protein expressed, Munc13-4 (Zhu, et al., in press).

My laboratory also has substantial collaborative relationships with others in the CF Center, most of which are centered on 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.  Of particular interest is a project involving patients with primary ciliary dyskinesia (PCD), a mucociliary clearance disease resulting from genetic abnormalities in the ciliary axoneme that cause the cilia to be dysfunctional.  We have developed a quantitative empirical model describing ciliary shape dynamics during the beat cycle, and are now comparing it with the dynamics observed in PCD cilia, from patients with different genetic abnormalities.  These projects all involve quantitative conventional and confocal microscopy, and/or molecular techniques.

Bill Davis holds a joint appointment in Pulmonary Medicine and his laboratory is located within the Cystic Fibrosis/Pulmonary Research & Treatment Center. You may send electronic mail to Dr. Davis at: cwdavis@med.unc.edu

Selected Recent Publications:

Davis, C.W. and B.F. Dickey.  Regulated airway goblet cell mucin secretion.  Ann. Rev. Physiol. In Press. 

Zhu, Y., C. Ehre, Abdullah, L.H.,  J.K. Sheehan, M. Roy, C.M. Evans, B.F. Dickey, and C.W. Davis In Press.  MUNC13-2-/- Baseline Secretion Defect Reveals Source of Oligomeric Mucins in Mouse Airways.   J. Physiol.

Ehre, C.,  Y. Zhu,  L.H. Abdullah, J. Olsen,  K.I. Nakayama, K. Nakayama,  R.O. Messing, and C.W.  Davis.  2007.   PKCε, a P2Y2-R downstream effector in regulated mucin secretion from airway goblet cells.  Am. J. Physiol., Cell. 293:C1445-C1454.

Abdullah, L.H. and C.W. Davis.  2007.  Regulation of Airway Goblet Cell Mucin Secretion by Tyrosine Phosphorylation Signaling Pathways.  Am. J. Physiol., Lung, 293:L591-L599.

Kreda, S.M. S.F. Okada, C.A. van Heusden, W. O’Neal, S. Gabriel, L.H. Abdullah, C.W. Davis, R.C. Boucher, and E.R. Lazarowski.  2007.  Coordinated release of nucleotides and mucin from  human airway Calu-3 cells.  J. Physiol.,   584:245-259.

Winters, S., C.W. Davis, and R.C. Boucher.  2007.  Mechanosensitivity of Mouse Tracheal Ciliary Beat Frequency:  Roles for Ca2+o, purinergic signaling, tonicity, and viscosity.  Am. J. Physiol.  292:L614-624.

Rossi, A.H., W.C. Salmon, M. Chua, and C.W. Davis.  2007.  Ca2+ signaling in human airway goblet cells following purinergic activation.  Am. J. Physiol. 292:L92-L98.

Ehre, C., A.H. Rossi, L.H. Abdullah, K. De Pestel, S. Hill, J.C. Olsen, and C.W. Davis.  2005.  Barrier Role of Actin Filaments in Regulated Mucin Secretion from Airway Goblet Cells.  Am. J. Physiol. 288:C46-C56.

Holmén, J.,  N.G. Karlsson, LH. Abdullah, S.H. Randell, J.K. Sheehan, G.C.  Hansson, and C.W. Davis.   2004.  Mucins and their O-Glycans from Normal and Cystic Fibrosis Bronchial Epithelial Cell Cultures.  Am. J. Physiol. 287:L824-L834.