Silvia M Kreda, PhD

Research Focus

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.

Mucin secretion studies

We have optimized in vitro and in vivo models of lung diseases for our mucin production studies. Preferred in vitro models are based on primary cultures of airway epithelial cells (HBECs). These cell cultures are derived from human lung tissues, and constitute a near physiologic epithelium. For example, using HBECs, we have recently identified serine proteases, acting through G protein-coupled protease activated receptors (PARs), as potent secretagogues of mucin granules in airway epithelium. Most serine proteases are at low levels in the normal lung, but are abundant during inflammation in obstructive lung diseases. Thus, PAR stimulation is a relevant regulatory pathway of mucin secretion in inflammatory airway diseases.

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.

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.

 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.

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).

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.

 

Collaborations

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.

Biosketch

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.

Education

  • 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

Funding Sources

Cystic Fibrosis Foundation, National Institutes of Health (NIH), NCTraCS (the NIH CTSA at UNC-CH), AstraZeneca, and Forest Research Institute.

CF Walk Large

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.

  

Publications

Click here to be taken to current publications or please see the Pubmed feed in the righthand column.

[top]

Journal Covers

[top]

Laboratory Personnel