Human airway trypsin-like protease induces mucin5AC hypersecretion via a protease-activated receptor 2-mediated pathway in human airway epithelial cells
ABSTRACT Mucus hypersecretion is a common feature in chronic airway diseases, and serine proteases play a critical role in this process. However, the mechanisms by which serine proteases induce mucin5AC hypersecretion have not been fully explored. In this study, we characterized human airway trypsin-like protease (HAT), a serine protease that is found in the mucoid sputum of patients with chronic airway diseases and is an agonist of protease-activated receptor 2 (PAR2)-induced cellular responses in human bronchial epithelial cells (16HBE). We also investigated the potential involvement of PAR2 in this process. We found that both HAT and PAR2-AP enhance the exocytosis of mucin5AC protein, whereas HAT, but not PAR2-AP, enhances the expression of mucin5AC mRNA. PAR2 is expressed at a much higher level in the cells than the other three PARs. Transfection with an siRNA against the PAR2 receptor or Gαq/11 protein or pretreatment with the Gαq/11 protein inhibitor YM-254890, the PLC inhibitor U73122 or the intracellular Ca(2+) chelator BAPTA-AM all effectively attenuated the HAT-induced cellular responses. Taken together, these results indicate that HAT can stimulate mucin5AC hypersecretion through a PAR2-mdiated signaling pathway in 16HBE cells. Thus, PAR2 could represent a novel therapeutic target for chronic airway diseases with mucus hypersecretion.
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ABSTRACT: Protease-activated receptors (PARs) are a family of seven transmembrane, G-protein-coupled receptors which are activated by multiple serine proteases through specific N-terminal proteolytic cleavage and the unmasking of a tethered ligand. The majority of PAR activating proteases described to date are soluble proteases that are active during injury, coagulation and inflammation. Less investigation however, has focused on the potential for membrane-anchored serine proteases to regulate PAR activation. Testisin is a unique trypsin-like serine protease that is tethered to the extracellular membrane of cells through a glycophosphatidylinositol (GPI)-anchor. Here we show that the N-terminal domain of PAR-2 is a substrate for testisin, and that proteolytic cleavage of PAR-2 by recombinant testisin activates downstream signaling pathways including intracellular calcium mobilization and ERK1/2 phosphorylation. When testisin and PAR-2 are co-expressed in HeLa cells, GPI-anchored testisin specifically releases the PAR-2 tethered ligand. Conversely, knockdown of endogenous testisin in NCI/ADR-Res ovarian tumor cells reduces PAR-2 N-terminal proteolytic cleavage. The cleavage of PAR-2 by testisin induces activation of intracellular serum response element (SRE) and NFkappaB signaling pathways, and the induction of IL-8 and IL-6 cytokine gene expression. Furthermore, the activation of PAR-2 by testisin results in the loss and internalization of PAR-2 from the cell surface. This study reveals a new biological substrate for testisin, and is the first demonstration of the activation of a PAR by a serine protease GPI-linked to the cell surface. Copyright © 2014, The American Society for Biochemistry and Molecular Biology.Journal of Biological Chemistry 12/2014; DOI:10.1074/jbc.M114.628560 · 4.60 Impact Factor
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ABSTRACT: Secretory epithelial cells of the proximal airways synthesize and secrete gel-forming polymeric mucins. The secreted mucins adsorb water to form mucus that is propelled by neighboring ciliated cells, providing a mobile barrier which removes inhaled particles and pathogens from the lungs. Several features of the intracellular trafficking of mucins make the airway secretory cell an interesting comparator for the cell biology of regulated exocytosis. Polymeric mucins are exceedingly large molecules (up to 3 × 10(6) Da per monomer) whose folding and initial polymerization in the ER requires the protein disulfide isomerase Agr2. In the Golgi, mucins further polymerize to form chains and possibly branched networks comprising more than 20 monomers. The large size of mucin polymers imposes constraints on their packaging into transport vesicles along the secretory pathway. Sugar side chains account for >70% of the mass of mucins, and their attachment to the protein core by O-glycosylation occurs in the Golgi. Mature polymeric mucins are stored in large secretory granules ∼1 μm in diameter. These are translocated to the apical membrane to be positioned for exocytosis by cooperative interactions among myristoylated alanine-rich C kinase substrate, cysteine string protein, heat shock protein 70, and the cytoskeleton. Mucin granules undergo exocytic fusion with the plasma membrane at a low basal rate and a high stimulated rate. Both rates are mediated by a regulated exocytic mechanism as indicated by phenotypes in both basal and stimulated secretion in mice lacking Munc13-2, a sensor of the second messengers calcium and diacylglycerol (DAG). Basal secretion is induced by low levels of activation of P2Y2 purinergic and A3 adenosine receptors by extracellular ATP released in paracrine fashion and its metabolite adenosine. Stimulated secretion is induced by high levels of the same ligands, and possibly by inflammatory mediators as well. Activated receptors are coupled to phospholipase C by Gq, resulting in the generation of DAG and of IP3 that releases calcium from apical ER. Stimulated secretion requires activation of the low affinity calcium sensor Synaptotagmin-2, while a corresponding high affinity calcium sensor in basal secretion is not known. The core exocytic machinery is comprised of the SNARE proteins VAMP8, SNAP23, and an unknown Syntaxin protein, together with the scaffolding protein Munc18b. Common and distinct features of this exocytic system in comparison to neuroendocrine cells and neurons are highlighted.Frontiers in Endocrinology 09/2013; 4:129. DOI:10.3389/fendo.2013.00129
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ABSTRACT: Mucus hypersecretion is a remarkable pathophysiological manifestation in airway obstructive diseases. These diseases are usually accompanied with elevated shear stress due to bronchoconstriction. Previous studies have reported that shear stress induces mucin5AC (MUC5AC) secretion via actin polymerization in cultured nasal epithelial cells. Furthermore, it is well known that cortactin, an actin binding protein, is a central mediator of actin polymerization. Therefore, we hypothesized that cortactin participates in MUC5AC hypersecretion induced by elevated shear stress via actin polymerization in cultured human airway epithelial cells. Compared with the relevant control groups, Src phosphorylation, cortactin phosphorylation, actin polymerization and MUC5AC secretion were significantly increased after exposure to elevated shear stress. Similar effects were found when pretreating the cells with jasplakinolide, and transfecting with wild-type cortactin. However, these effects were significantly attenuated by pretreating with Src inhibitor, cytochalasin D or transfecting cells with the specific small interfering RNA of cortactin. Collectively, these results suggest that elevated shear stress induces MUC5AC hypersecretion via tyrosine-phosphorylated cortactin-associated actin polymerization in cultured human airway epithelial cells.The international journal of biochemistry & cell biology 10/2013; DOI:10.1016/j.biocel.2013.09.013 · 4.24 Impact Factor