Role of Protein Kinase C Delta in Airway Mucin Secretion
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Date
2006-11-19
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Abstract
Mucin hypersecretion is a prominent pathophysiologic feature of chronic airway diseases such as cystic fibrosis, asthma and chronic bronchitis. Exacerbated mucin secretion can lead to impaired mucociliary function, increased susceptibility to bacterial pathogens, potentiation of inflammatory responses, and, in extreme cases, death. Despite these deleterious effects, effective therapy to alleviate mucin hypersecretion remains to be developed.
The intention of this work was to elucidate the mechanism of mucin secretion in response to secretagogues utilizing normal human bronchial epithelial (NHBE) cells in vitro. NHBE cells were maintained in air/liquid interface and allowed to differentiate into a normal airway epithelium containing mucin secreting (goblet), ciliated and basal cells. Mucin secretion was measured by an enzyme linked immunoabsorbent assay (ELISA) using antibodies recognizing specific mucin proteins (MUC5AC, MUC5B and MUC2) or pan-secreted mucins.
In manuscript one, we elucidate some aspects of the mechanism whereby human neutrophil elastase (HNE) enhances mucin secretion from NHBE cells. We found that HNE provokes mucin secretion in a concentration-dependent manner, with maximal stimulation (more than two-fold) occurring within a short (15 minute) time period. The mucins, MUC5AC and MUC5B, but not MUC2, were released in response to 500nM HNE. Myristoylated alanine-rich C-kinase substrate (MARCKS) is rapidly phosphorylated in response to HNE exposure (within 2 minutes), suggesting activation of protein kinase C (PKC) is involved in HNE-induced mucin hypersecretion. PKCδ was the only isoform to translocate from cytoplasm to membrane in response to HNE, and inhibition of PKCδ by a specific pharmacologic inhibitor (rottlerin) attenuated HNE-mediated mucin secretion. Therefore, we concluded that HNE induces mucin secretion via the activation of PKCδ in normal human bronchial epithelial cells in vitro.
In manuscript two, we further investigated the role of PKCδ in mucin secretion. Selective activation of PKCδ by bryostatin 1 provoked mucin secretion and induced phosphorylation of MARCKS in NHBE cells. Suppression of PKCδ by rottlerin significantly attenuated HNE-or PMA-mediated mucin secretion as well as phosphorylation of MARCKS. A virally immortalized human bronchial epithelial cell line (HBE-1) was utilized for transient transfections. Transfection of HBE-1 cells with a dominant-negative PKCδ construct (pEGFP-N1/PKCδK376R) significantly attenuated PMA-induced mucin secretion and phosphorylation of MARCKS compared to cells transfected with empty vector (pEGFP-N1) alone. These results suggest that PKCδ regulates mucin hypersecretion in human airway epithelial cells.
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protein kinase c delta, mucin secretion
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Degree
PhD
Discipline
Toxicology
