The overall objective of our research is to identify signaling molecules/pathways that regulate mucin secretion in airway goblet cells. A major goal of these studies is to uncover potential targets of clinical interest to control abnormal lumenal mucus accumulation in lung diseases.
Mucus and mucins
Airway epithelia are covered by a thin liquid film of mucus, which is essential for epithelial homeostasis and lung defense. Mucus is a complex aqueous solution containing electrolytes and macromolecules, produced and maintained by the underlying epithelium. Gel-forming mucins, secreted via exocytosis by mucous/goblet cells, are the main macromolecular component of airway mucus and impart the physicochemical characteristics to mucus in physiological and pathological conditions. In chronic obstructive lung diseases (e.g., CF, COPD, asthma), a key consequence of epithelial inflammation is goblet cell or mucous hyperplasia. In this state, the airway surface epithelium and glands are dominated by goblet cells and mucin secretion is greatly increased. Mucin hypersecretion produces stasis and accumulation of mucus on airway surfaces. Further, mucus plaques incorporate cell debris, DNA, and other macromolecules, generating a tenacious mucus that blocks the airway lumen and is difficult to expectorate. Mucus plugging elicits further airway inflammation leading to a vicious cycle of disease in the lung. For example, fatal asthmatic airways almost always exhibit complete lumenal obstruction with mucus, while mucus plaques in CF are a main vehicle for chronic infections leading to lethal loss of lung tissue.
Mucus plaques in obstructive lung diseases. Bronchial tissues from normal donors and CF individuals were processed and stained with H&E. Normal surface epithelium is dominated by ciliated cells, only few goblet cells are present, and the lumen is limpid. In contrast, CF surface epithelium is dominated by goblet cells (mucous hyperplasia), inflammation is noticeable in the tissues underneath the epithelium, and the lumen is filled with a tethered mucus plaque containing bacteria, inflammatory cells, and cell debris. Magnification is the same for both images.
Mucin secretion studies
|Primary bronchial epithelial cultures secret mucins in response to PAR agonists. Confocal microscopy image of HBECs revealing that MUC5AC (red) is present within granules in goblet cells (arrow), while CFTR (green) is localized at the apical membrane of ciliated cells (arrowheads); bar, 10 µm (Kreda et al, 2005 Mol Biol Cell 16, 2154). For detailed immunostaining protocols see Kreda et al, Methods Mol Biol. 2011; 742:15-33. Primary bronchial epithelial cultures secrete mucin in response to stimulation with PAR agonist thrombin, PAR1peptide, and PAR2peptide; P<0.01 (Kreda et al., 2010 J Physiol 588: 2255; with permission by Wyley-Blackwell).|
In addition to using HBECs for in vitro studies, we have characterized Calu-3 cells as an airway epithelial goblet cell model. Calu-3 cell cultures are composed by two cell types: ~60% of the cells express CFTR while ~40% express goblet cell mucin granules.Calu-3 cells express biological features of native goblet cells even when cultured on simple plastic supports, a characteristic that can be exploited in high-throughput assays for mucin production.
|Calu-3 cells produce mucin granules. Left, Electron microscopy images of Calu-3 cells displaying dense-core mucin granules; the middle image is an amplification of the red rectangle. Right, Immunostaining of MUC5AC granules (green) in Calu-3 cells; nuclei are stained red. Modified from Kreda et al, J Physiol 2007, 584:245 with permission by Wyley-Blackwell.|
Using a combination of molecular biology, biochemical, and fluorescence-based confocal microscopy techniques, our studies in HBECs and Calu-3 cells revealed that regulation of mucin exocytosis via PAR activation involves intracellular calcium mobilization, cytoskeleton remodeling, and activation of Rho and myosin light chain kinases. Currently, we are identifying other signaling elements necessary for mucin exocytosis that are recruited downstream from G-protein coupled receptor activation. Our findings on regulatory elements of mucin exocytosis in cell cultures are being further tested in vivo in mouse models of lung diseases in collaboration with the UNC-CF Center Mouse Models Core.
|Real-time exocytosis studies reveal that PAR agonists stimulate secretion of mucin granules. A- Mucin granules labeled with MUC5AC antibodies in fixed airway epithelia (top) and FM 4-64 in live HBECs (bottom); yellow line shows ~position of DIC/FM 4-64 image; bars, 10µm; CC=ciliated cells, GC= goblet cells; published in Kreda et al, 2008 Physiology News 71:19-21. B, C- Real-time exocytosis of mucin granules was studied in Calu-3 cells loaded with quinacrine in response to PAR agonists thrombin, PAR1P, or PAR2P (control = vehicle). B- Overlay of the DIC and fluorescence confocal images of quinacrine-labeled Calu-3 cells; bar, 10 um. C- Cumulative changes in fluorescence intensity associated with mucin granules after 5 min of agonist challenge; P<0.01 (for details, Kreda et al., 2010 J Physiol 588:2255).|
We have optimized a protocol for isolation/purification of intact mucin granules (Kreda et al, 2010 J Physiol 588:2255). In collaboration with Dr. Lazarowsk’s lab, we established that mucin granules contain a pool of nucleotides that is coordinately released with mucins during granule exocytosis. This mechanism of regulated nucleotide release provides paracrine signaling to ciliated cells for mucin hydration and dispersion into the airway surface mucus.
|Model of adenyl nucleotide regulation in airway epithelia. The schematics represent a ciliated and goblet cell of the airway surface epithelium bathed in the surface mucus layer (ASL). Exocytosis of mucin is accompanied by release of adenyl nucleotides present in mucin granules as co-cargo molecules. In the mucus layer, nucleotides (NTPs) are rapidly metabolized by ecto-nucleotidases into adenosine. Adenyl purines have autocrine and paracrine regulatory activities on epithelial cells. For example, adenosine stimulates the A2b receptor on ciliated cells. CFTR, which is expressed in ciliated cells, is activated by A2b receptor-promoted cAMP formation (and PKA activation, not shown). Thus, chloride secretion is increased and sodium absorption is reduced (by CFTR-mediated inhibition of ENaC), which generates the driving gradient for water secretion necessary to disperse newly secreted mucins into the ASL. ATP released from mucin granules stimulates P2Y2 receptors on goblet cells for further mucin secretion, and on ciliated cells resulting in activation of CACC (calcium activated chloride channel), activation of CFTR (via PKC), and inhibition of ENaC.|
The isolation of mucin granules has also allowed us to characterize components of the airway mucin granule exocytotic machinery. Recently, we identified VAMP8 as a critical SNARE for mucin granule exocytosis in airway goblet cells of human airway epithelia (Jones et al, 2012 J Physiol 590:545).
|VAMP8 is associated with mucin granules in HBECs and Calu-3 cells. Confocal microscopy images of immunostaining of VAMP8 and MUC5AC in HBECs and Calu-3 cells. Both proteins localize in ~ 1µm-diameter mucin granules in baseline conditions in HBE (A) and Calu-3 (B) cells, but not after agonist stimulation in Calu-3 cells (C). Bar, 10 µm (published in Kreda et al, 2010 J Physiol 588:2255 and Jones et al, 2012 J Physiol 590:545; with permission by Wyley-Blackwell.)|
The regulatory role of VAMP8 in airway goblet cell exocytosis was further investigated in vivo. As observed in in vitro studies, in VAMP8 knockout mice with induced airway inflammation, agonist-stimulated mucin degranulation is inhibited in airway epithelium.
|Mucin secretion is reduced in IL-13-treated VAMP8 KO mice. A- Airway goblet cell degranulation in IL-13-treated mice was assessed by incubating tracheas ex vivo with vehicle or ATP, fixed, and stained with AB-PAS. Left, images arerepresentative longitudinal views of KO, HETerozygous, and WT mouse tracheas; dark purple-blue staining indicates AB-PAS positive substances (i.e., mucin); tracheas from age matched mice with no IL-13 treatment (naïve) are shown for comparison; right, AB-PAS staining quantification. B- Western blot of mouse BALs using an antibody against mouse MUC5B; arrowhead indicates the position of MUC5B; right, western blot quantification; mean ± SEM, * = p<0.01. For more details, Jones J Physiol 590:545.|
Our lab has developed/optimized in vitro and in vivo models of lung diseases and a battery of quantitative assays to evaluate mucin production in airway epithelia. We have numerous successful collaborations with scientists from UNC and other Institutions.
In collaboration with pharmaceutical companies, we have been studying the effect of novel drugs on mucin production and their potential therapeutic use in obstructive lung diseases. Information about future/potential collaborations can be obtained by directly contacting Silvia Kreda.
Silvia M. Kreda, PhD, Assistant Professor of Medicine holds an appointment in the Department of Medicine. Her laboratory is located within the Cystic Fibrosis/Pulmonary Research & Treatment Center.
- 1992 Post Doctorate: University of North Carolina at Chapel Hill
- 1992 Ph.D.: University of Buenos Aires, Argentina
- 1987 M.S.: University of Buenos Aires, Argentina
- 1986 Pharm D., R.Ph.: University of Buenos Aires, Argentina
- 1984 B.S.: University of Buenos Aires, Argentina
Cystic Fibrosis Foundation, National Institutes of Health (NIH), NCTraCS (the NIH CTSA at UNC-CH), AstraZeneca, and Forest Research Institute.
Silvia (front, kneeling) walking with her certified therapy dog, Whiskey, and CF Center colleagues during the annual Chapel Hill Great Strides walk to raise awareness and funds for Cystic Fibrosis research.
|Kreda et al, 2001||Kreda et al, 2004||Kreda et al, 2005|
|Okada et al, 2006||Kreda et al, 2007||Mall et al, 2008|
|Kreda et al, 2008|
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