nPKCε, a P2Y₂-R downstream effector in regulated mucin secretion from airway goblet cells

Abstract

Ehre C, Zhu Y, Abdullah LH, Olsen J, Nakayama KI, Nakayama K, Messing RO, Davis CW. nPKCε, a P2Y 2-R downstream effector in regulated mucin secretion from airway goblet cells. Am J Physiol Cell Physiol 293: C1445-C1454, 2007. First published August 29, 2007; doi:10.1152/ajpcell.00051.2007.-Airway goblet cell mucin secretion is controlled by agonist activation of P2Y 2 purinoceptors, acting through Gq/PLC, inositol-1,4,5-trisphosphate (IP 3), diacylglycerol, Ca 2ϩ and protein kinase C (PKC). Previously, we showed that SPOC1 cells express cPKC␣, nPKC␦, nPKCε, and nPKC; of these, only nPKC␦ translocated to the membrane in correlation with mucin secretion (Abdullah LH, Bundy JT, Ehre C, Davis CW. Am J Physiol Lung Physiol 285: L149 -L160, 2003). We have verified these results and pursued the identity of the PKC effector isoform by testing the effects of altered PKC expression on regulated mucin release using SPOC1 cell and mouse models. SPOC1 cells overexpressing cPKC␣, nPKC␦, and nPKC had the same levels of ATP␥S-and phorbol-1,2-myristate-13-acetate (PMA)-stimulated mucin secretion as the levels in empty retroviral vector expressing cells. Secretagogue-induced mucin secretion was elevated only in cells overexpressing nPKCε (14.6 and 23.5%, for ATP␥S and PMA). Similarly, only SPOC1 cells infected with a kinase-deficient nPKCε exhibited the expected diminution of stimulated mucin secretion, relative to wild-type (WT) isoform overexpression. ATP␥S-stimulated mucin secretion from isolated, perfused mouse tracheas was diminished in P2Y 2-R null mice by 82% relative to WT mice, demonstrating the utility of mouse models in studies of regulated mucin secretion. Littermate WT and nPKC␦ knockout (KO) mice had nearly identical levels of stimulated mucin secretion, whereas mucin release was nearly abolished in nPKCε KO mice relative to its WT littermates. We conclude that nPKCε is the effector isoform downstream of P2Y 2-R activation in the goblet cell secretory response. The translocation of nPKC␦ observed in activated cells is likely not related to mucin secretion but to some other aspect of goblet cell biology.protein kinase C; mucins; goblet cells; exocytosis; airways; epithelium; lung OLIGOMERIC MUCINS provide the molecular scaffolding of mucus (58), which in the lung is crucial for defense against inhaled particulates and pathogens by the mucociliary clearance system (34). In healthy subjects, oligomeric mucins are produced and secreted from goblet cells in the superficial epithelium of the airways and from submucosal glands (20), in quantities sufficient for normal mucociliary clearance. The conditions of infection and inflammation in obstructive lung disease (chronic bronchitis, emphysema, cystic fibrosis, asthma), however, drive metaplastic, hyperplasic, and hypertrophic processes, resulting in mucus hyperproduction and plugged airways. In humans and possibly other primates, innervation of the superficial epithelium is sparse and regulation of goblet cell secretion is effected primarily through local mediators (see Re...

Regulated Airway Goblet Cell Mucin Secretion

“…The use of retroviral infection vectors with SPOC1 cells, however, indicated that overexpression of cPKCα, nPKCδ, and nPKCη was without effect, whereas overexpression of nPKCε caused an increase in agonist-induced mucin secretion. Additionally, in perfused mouse tracheas the mucin secretory response to agonist was blunted in the nPKCε-deficient mouse, but not the nPKCδ-deficient mouse (48). Hence, nPKCε appears to be the isoform mediating agonist effects in goblet cells.…”

“…P2Y 2 -R was cloned from the airways (42) and appears to be the major purinoceptor regulating mucociliary clearance in the airways, including mucin release from goblet cells. ATP and UTP, the primary P2Y 2 -R agonists, elicit maximal secretion from goblet cells in all vertebrate species examined (e.g., see References 9 and 43), including human (44,45), the receptor mRNA has been identified in two goblet cell models (46,47), and the tracheal mucin secretory response to ATP is severely compromised in the P2Y 2 -R-deficient mouse (48).…”

Synaptotagmin 2 Couples Mucin Granule Exocytosis to Ca2+ Signaling from Endoplasmic Reticulum

“…These bind to epithelial apical P2Y 2 receptors that activate G q , which in turn activates phospholipase C␤ 1 generating the intracellular second messengers diacylglycerol and inositol trisphosphate (IP 3 ). 3 Diacylglycerol directly induces mucin granule exocytosis by activating the priming protein Munc13-2 (4), and indirectly regulates exocytosis by activating protein kinase C⑀ (5). IP 3 induces the release of Ca 2ϩ from intracellular stores, resulting in a rise in cytoplasmic Ca 2ϩ that rapidly triggers mucin granule exocytosis (6 -9).…”

Regulation of the Epithelial Na+ Channel by the RH Domain of G Protein-coupled Receptor Kinase, GRK2, and Gαq/11

“…These G␣ subunits share over 80% homology in their amino acid sequences (45), hence their ability to inhibit the channel might reflect a promiscuous capacity to activate the same downstream signaling pathways, one of which inhibits ENaC. Gq/11-coupled GPCRs are known to regulate several cellular events in epithelial cells, including intracellular Ca 2ϩ mobilization (46), mucin secretion (47,48), and anion secretion (49). Our data suggest that, Gq/11 plays a small, but significant, part in the P2Y 2 receptor-mediated signaling mechanism that inhibits activity of ENaC.…”