Abstract
Hypersecretion of airway mucin characterizes numerous respiratory diseases. Although diverse pathological stimuli can provoke exocytotic release of mucin from secretory cells of the airway epithelium, mechanisms involved remain obscure. This report describes a new paradigm for the intracellular signaling mechanism regulating airway mucin secretion. Direct evidence is provided that the myristoylated alanine-rich C kinase substrate (MARCKS) is a central regulatory molecule linking secretagogue stimulation at the cell surface to mucin granule release by differentiated normal human bronchial epithelial cells in vitro. Down-regulation of MARCKS expression or disruption of MARCKS function in these cells inhibits the secretory response to subsequent stimulation. The intracellular mechanism controlling this secretory process involves cooperative action of two separate protein kinases, protein kinase C and cGMP-dependent protein kinase. Upon stimulation, activated protein kinase C phosphorylates MARCKS, causing translocation of MARCKS from the plasma membrane to the cytoplasm, where it is then dephosphorylated by a protein phosphatase 2A that is activated by cGMP-dependent protein kinase, and associates with both actin and myosin. Dephosphorylated cytoplasmic MARCKS would also be free to interact with mucin granule membranes and thus could link granules to the contractile cytoskeleton, mediating their movement to the cell periphery and subsequent exocytosis. These findings suggest several novel intracellular targets for pharmacological intervention in disorders involving aberrant secretion of respiratory mucin and may relate to other lesions involving exocytosis of membrane-bound granules in various cells and tissues.
Highlights
Hypersecretion of airway mucin characterizes numerous respiratory diseases
Preliminary studies examining mucin secretion in response to phorbol 12-myristate 13-acetate (PMA) stimulation at various concentrations for different times indicated that activation of protein kinase C (PKC) alone did not induce significant mucin secretion from normal human bronchial epithelial (NHBE) cells, a moderate secretory response was repeatedly observed at PMA concentrations higher than 100 nM (0.05 Ͻ p Ͻ 0.1)
Our preliminary studies showed that UTP (100 M) could induce a significant increase in mucin secretion from NHBE cells after a 2-h exposure
Summary
Vol 276, No 44, Issue of November 2, pp. 40982–40990, 2001 Printed in U.S.A. MARCKS Protein Is a Key Molecule Regulating Mucin Secretion by Human Airway Epithelial Cells in Vitro*. Many of these mucin secretagogues are known to activate several protein kinases, and studies examining the regulation of excess secretion of mucin by airway epithelial cells from various species have consistently implicated involvement of either protein kinase C (PKC)1 [3,4,5,6] or cGMP-dependent protein kinase (PKG) [7] in the secretory process. We reported [8] development of a procedure to culture normal human bronchial epithelial (NHBE) cells in an air/liquid interface system in which the cells differentiate to a heterogeneous population containing secretory (goblet), ciliated, and basal cells that mimic their in vivo appearance and function These cell cultures provide an ideal in vitro model system to study mechanisms regulating mucin secretion from human airway epithelium. MARCKS interacts with actin and myosin in the cytoplasm and could tether the granules to the cellular contractile apparatus, mediating subsequent granule movement and exocytosis
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