Protease-activated receptor-2 (PAR-2) is a weak enhancer of mucin secretion by human bronchial epithelial cells in vitro

PAR-2, a member of a family of G-protein-coupled receptors, can be activated by serine proteases via proteolytic cleavage. PAR-2 expression is known to be upregulated in respiratory epithelium subsequent to inflammation in asthma and chronic obstructive pulmonary disease (COPD). Since these diseases also are characterized by excessive mucus production and secretion, we investigated whether PAR-2 could be linked to mucin hypersecretion by airway epithelium. Normal human bronchial epithelial (NHBE) cells in primary culture or the human bronchial epithelial cell lines, NCI-H292 and HBE-1, were used. NHBE, NCI-H292, and HBE-1 cells expressed prominent levels of PAR-2 protein. Short-term (30min) exposure of cells to the synthetic PAR-2 agonist peptide (SLIGKV-NH2) elicited a small but statistically significant increase in mucin secretion at high concentrations (100microM and 1000microM), compared to a control peptide with reversed amino acid sequence (VKGILS-NH2). Neither human lung tryptase nor bovine pancreatic trypsin, both PAR-2 agonists, affected NHBE cell mucin secretion when added over a range of concentrations. Knockdown of PAR-2 expression by siRNA blocked the stimulatory effect of the AP. The results suggest that, since PAR-2 activation only weakly increases mucin secretion by human airway epithelial cells in vitro, PAR-2 probably is not a significant contributor to mucin hypersecretion in inflamed airways.     

Type IV collagen reduces mucin 5AC secretion in three-dimensional cultured human primary airway epithelial cells

Mucin 5AC (MUC5AC) hypersecretion induces airway narrowing in patients with asthma, which leads to breathing problems. We investigated the regulation of MUC5AC secretion by extracellular matrix (ECM) proteins in human primary airway epithelial cells from patients with asthma. The addition of type IV collagen to three-dimensional cultured human primary airway epithelial cells, which mimics the airway surface, reduced MUC5AC secretion in the medium, while the addition of laminin increased MUC5AC secretion. Furthermore, the addition of fibronectin did not affect MUC5AC secretion. In particular, the repeated addition of a low concentration of type IV collagen demonstrated a cumulative effect on the reduction in MUC5AC secretion. Human primary cells incubated with type IV collagen showed downregulated extracellular signal-regulated kinase (ERK) activity, which induced MUC5AC hypersecretion but did not affect Akt activity. These results suggest that the addition of type IV collagen to the apical surface of primary cells downregulates MUC5AC secretion and has a cumulative effect on MUC5AC secretion which might be effected via the ERK signaling pathway.     

Protein kinase-A-mediated secretion of mucin from human colonic epithelial cells

Both Ca(2+)- and cAMP-mediated second messenger cascades acutely regulate mucin secretion from human colonic epithelial cells. To better understand the cAMP-dependent regulation of mucin secretion we have characterized the complement of cAMP-dependent protein kinase (PKA) isoforms in mucus-secreting T84 cells, and determined which of these isoforms is responsible for agonist-stimulated mucin secretion. Our results show the presence of both type I and type II PKA in cells that also contain large mucin granules. Forskolin caused a rapid and sustained increase in PKA activity that reached a maximum 5-10 min following its addition. Secretion of mucin was detected 15 min following exposure to forskolin, and continued to increase for a further 15 min before reaching a plateau. Mucin secretion was also measured in the presence of combinations of site-selective cAMP analog pairs, which preferentially activate either type I or type II PKA. Similar levels of mucin secretion were observed for both type I and type II PKA-selective analog pairs. Subsequent addition of forskolin was unable to further increase mucin secretion. Thus, activation of either type I or type II PKA is able to maximally stimulate secretion of mucins from T84 human colonic epithelial cells.     

Progesterone in vitro stimulates secretion of a specific protein by ovine epithelial endometrial cells

Cultured ovine epithelial endometrial cells from oestrogen-treated ovariectomized ewes were treated in vitro with combinations of oestradiol-17 beta (E), progesterone (P) and the P-receptor antagonist RU486 (each 10(-6) to 10(-9) M), in the presence of [35S]methionine. Neither DNA content of dishes nor total protein were increased in treatment compared with control dishes. Incorporation of [35S] into secreted protein was lower from cells treated in vitro with P or E + P (10(-9) M) than from those treated with E (10(-9) M, P less than 0.01). Incorporation of [35S] into cellular protein was decreased by P (10(-9) M, P less than 0.025). SDS-PAGE analysis of secreted proteins enabled measurement of levels of a 46K protein which is secreted maximally following E + P administration in vivo. In vitro, P either alone or with E (each 10(-7) M) increased the abundance of the 46K protein in cell secretions by a factor of 1.5 +/- 0.1 (N = 9) or 1.8 +/- 0.3 (N = 10) respectively (P less than 0.01) compared with controls. The administration of E (10(-7) M) or either or both steroids at 10(-9) M, was without effect. RU486 alone (10(-6) to 10(-8) M) was also without effect but in the presence of E + P or P, blocked the increase in the 46K protein, suggesting this effect is mediated via binding of P to its receptor.     

Transient receptor potential vanilloid 1 receptors mediate acid-induced mucin secretion via Ca2+ influx in human airway epithelial cells

Mucin hypersecretion is a key pathological feature of inflammatory respiratory diseases. Previous studies have reported that acids (gastroesophageal reflux or environmental exposure) induce many respiratory symptoms and are implicated in the pathophysiology of obstructive airway diseases. To understand these mechanisms, we measured acid-induced mucin secretion in human bronchial epithelial cells. In the present study, acid induced inward currents of transient receptor potential vanilloid (TRPV)1 and mucin 5AC (MUC5AC) secretion dose dependently, which were inhibited by TRPV1 antagonist capsazepine in a concentration-dependent manner. TRPV1 agonist capsaicin mediated a concentration-dependent increase in TRPV1 inward currents and MUC5AC secretion. Furthermore, capsaicin enhanced acid-induced TRPV1 inward currents and MUC5AC secretion. Acid-induced Ca(2+) influx was prevented by capsazepine dose dependently and enhanced by capsaicin. Pretreatment only with capsaicin also increased the Ca(2+) concentration in a concentration-dependent manner. These data suggest that pharmacological inhibition of calcium-permeable TRPV1 receptors could be used to prevent acid-induced mucin secretion, thereby providing a potential mechanism to reduce their toxicity.     

Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells

Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO-3 by a Cl-offdependent, serosal Na+-dependent, serosal bumetanide-insensitive, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca2+-activated K+ channels, reduced HCO-3 secretion and caused the secretion of Cl- by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K+ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K+ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO-3 secretion to Cl- secretion because the uptake of HCO-3 across the basolateral membrane is mediated by a 4,4 '-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive Na+:HCO-3 cotransporter. Since the stoichiometry reported for Na+:HCO-3 cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO-3 entry and favor the secretion of Cl-. Therefore, differential regulation of the basolateral membrane K+ conductance by secretory agonists could provide a means of stimulating HCO-3 and Cl- secretion. In this context, cystic fibrosis transmembrane conductance regulator could serve as both a HCO-3 and a Cl- channel, mediating the apical membrane exit of either anion depending on basolateral membrane anion entry mechanisms and the driving forces that prevail. If these results with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO-3 secretion in the human airways warrants greater attention.     

Regulation of mucin secretion from human bronchial epithelial cells grown in murine hosted xenografts

Studies of regulated mucin secretion from goblet cells in primary cultures of human bronchial epithelial (HBE) cells have suffered, generally, from poor signal-to-noise ratios, with reported secretory responses of <100% (less than onefold) relative to baseline. Using, instead, HBE cells grown as xenografts in the backs of nude mice, we found that UTP (100 micro M) stimulated strong mucin secretory responses from isolated, luminally perfused preparations. The peak response (10 min) for 11 control experiments (37 xenografts) was 3.3 +/- 0.05-fold relative to baseline, and the time-integrated response (60 min) was 23.4 +/- 0.5-fold. Because responses to ATP and UTP were approximately equal, an apical membrane P2Y(2)-receptor (R) is suggested. Additionally, ADP activated mucin release from HBE xenografts, whereas UDP and 2-methlythio-ADP did not, a pattern of response inconsistent with known purinoceptors. Hence, either a novel receptor to ADP is suggested or there is significant conversion of ADP to ATP by ecto-adenylate kinase activity. Adenosine and a nitric oxide donor were without effect. Consistent with P2Y(2)-R coupling to phospholipase C, HBE xenografts responded to ionomycin and PMA; however, they were recalcitrant to forskolin and chlorophenylthio-cAMP, and to 8-bromo-cGMP. Hence, human airway goblet cells, like those of other species, appear to be regulated primarily via phospholipase C pathways, activated particularly by apical membrane P2Y(2)-R agonists.     

DIP (mDia interacting protein) is a key molecule regulating Rho and Rac in a Src-dependent manner

Cell movement is driven by the coordinated regulation of cytoskeletal reorganization through Rho GTPases downstream of integrin and growth-factor receptor signaling. We have reported that mDia, a target protein of Rho, interacts with Src and DIP. Here we show that DIP binds to p190RhoGAP and Vav2, and that DIP is phosphorylated by Src and mediates the phosphorylation of p190RhoGAP and Vav2 upon EGF stimulation. When endogenous DIP was inhibited by expressing dominant-negative mutants of DIP or siRNA, phosphorylation of p190RhoGAP and Vav2 upon EGF stimulation was diminished, and EGF-induced actin organization, distribution of p190RhoGAP and Vav2, and cell movement were affected. Therefore, DIP seems to transfer the complex of the three proteins from cytosol to beneath the membrane, and the three proteins, in turn, can be phosphorylated by Src. DIP inactivated Rho and activated Rac following EGF stimulation in the membrane fraction. Thus, DIP acts as a regulatory molecule causing Src kinase-dependent feedback modulation of Rho GTPases downstream of Rho-mDia upon EGF stimulation, and plays an important role in cell motility.     

Cortactin mediates elevated shear stress-induced mucin hypersecretion via actin polymerization in human airway epithelial cells

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.     

SNAP23 is selectively expressed in airway secretory cells and mediates baseline and stimulated mucin secretion

Airway mucin secretion is important pathophysiologically and as a model of polarized epithelial regulated exocytosis. We find the trafficking protein, SNAP23 (23-kDa paralogue of synaptosome-associated protein of 25 kDa), selectively expressed in secretory cells compared with ciliated and basal cells of airway epithelium by immunohistochemistry and FACS, suggesting that SNAP23 functions in regulated but not constitutive epithelial secretion. Heterozygous SNAP23 deletant mutant mice show spontaneous accumulation of intracellular mucin, indicating a defect in baseline secretion. However mucins are released from perfused tracheas of mutant and wild-type (WT) mice at the same rate, suggesting that increased intracellular stores balance reduced release efficiency to yield a fully compensated baseline steady state. In contrast, acute stimulated release of intracellular mucin from mutant mice is impaired whether measured by a static imaging assay 5 min after exposure to the secretagogue ATP or by kinetic analysis of mucins released from perfused tracheas during the first 10 min of ATP exposure. Together, these data indicate that increased intracellular stores cannot fully compensate for the defect in release efficiency during intense stimulation. The lungs of mutant mice develop normally and clear bacteria and instilled polystyrene beads comparable to WT mice, consistent with these functions depending on baseline secretion that is fully compensated.     

Mechanophysical stimulations of mucin secretion in cultures of nasal epithelial cells

Nasal epithelial cells secret mucins and are exposed in vivo to airflow-induced mechanophysical stresses, including wall shear stress (WSS), temperature, and humidity. In this work, human nasal epithelial cells cultured under air-liquid interface conditions were subjected to fields of airflow-induced oscillatory WSS at different temperature and humidity conditions. Changes in mucin secretion due to WSS were measured and the role of the cytoskeleton in mucin secretion was explored. Mucin secretion significantly increased in response to WSS in a magnitude-dependent manner with respect to static cultures and independently of the airflow temperature and humidity. In static cultures, mucin secretion decreased at high humidity with or without elevation of the temperature with respect to cultures at a comfortable climate. In cultures exposed to WSS, mucin secretion increased at high temperature with respect to cultures at comfortable climate conditions. The polymerization of actin microfilaments was shown to increase mucin secretion under WSS, whereas the dynamics of microtubule polymerization did not affect secretion. In conclusion, the data in this study show that mucin secretion is sensitive to oscillatory WSS as well as high temperature and humidity conditions.     

Effects of the cationic protein poly-L-arginine on airway epithelial cells in vitro

BACKGROUND: Allergic asthma is associated with an increased number of eosinophils in the airway wall. Eosinophils secrete cationic proteins, particularly major basic protein (MBP). AIM: To investigate the effect of synthetic cationic polypeptides such as poly-L-arginine, which can mimic the effect of MBP, on airway epithelial cells. METHODS: Cultured airway epithelial cells were exposed to poly-L-arginine, and effects were determined by light and electron microscopy. RESULTS: Poly-L-arginine induced apoptosis and necrosis. Transmission electron microscopy showed mitochondrial damage and changes in the nucleus. The tight junctions were damaged, as evidenced by penetration of lanthanum. Scanning electron microscopy showed a damaged cell membrane with many pores. Microanalysis showed a significant decrease in the cellular content of magnesium, phosphorus, sodium, potassium and chlorine, and an increase in calcium. Plakoglobin immunoreactivity in the cell membrane was decreased, indicating a decrease in the number of desmosomes CONCLUSIONS: The results point to poly-L-arginine induced membrane damage, resulting in increased permeability, loss of cell-cell contacts and generalized cell damage.     

Effect of floating a gel matrix on mucin release in cultured airway epithelial cells

Confluent cultures of primary hamster tracheal surface epithelial (HTSE) cells grown on a thick collagen gel are highly enriched with secretory cells and constitutively release mucins. In the present experiment, we examined the possible effect of mechanical strain of cultured HTSE cells on the release of mucin. The mechanical strain of cells was accomplished by several methods: 1) by floating the gel from the culture dish by rimming; 2) by treatment with EGTA which interrupts intercellular tight junctions; 3) by treatment with collagenase which disrupts the cell-matrix adhesion; and 4) by mechanically flexing the collagen gel matrix. All these conditions caused increases of mucin release without damage on the plasma membrane. We conclude that a number of mechanical strains which might alter cell shape can stimulate mucin release from cultured HTSE cells. Such a mechanism might be operative in the physiological regulation of airway goblet cell mucin secretion where mechanical strains may be induced on epithelial cells by underlying smooth muscles. © 1993 Wiley-Liss, Inc.     

Cystic fibrosis and the relationship between mucin and chloride secretion by cultures of human airway gland mucous cells

We investigated how cystic fibrosis (CF) alters the relationship between Cl(-) and mucin secretion in cultures of non-CF and CF human tracheobronchial gland mucous (HTGM and CFTGM, respectively) cells. Biochemical studies showed that HTMG cells secreted typical airway mucins, and immunohistochemical studies showed that these cells expressed MUC1, MUC4, MUC5B, MUC8, MUC13, MUC16, and MUC20. Effects of cumulative doses of methacholine (MCh), phenylephrine (Phe), isoproterenol (Iso), and ATP on mucin and Cl(-) secretion were studied on HTGM and CFTGM cultures. Baseline mucin secretion was not significantly altered in CFTGM cells, and the increases in mucin secretion induced by mediators were unaltered (Iso, Phe) or slightly decreased (MCh, ATP). Across mediators, there was no correlation between the maximal increases in Cl(-) secretion and mucin secretion. In HTGM cells, the Cl(-) channel blocker, diphenylamine-2-carboxylic acid, greatly inhibited Cl(-) secretion but did not alter mucin release. In HTGM cells, mediators (10(-5) M) increased mucin secretion in the rank order ATP > Phe = Iso > MCh. They increased Cl(-) secretion in the sequence ATP > MCh ≈ Iso > Phe. The responses in Cl(-) secretion to MCh, ATP, and Phe were unaltered by CF, but the response to Iso was greatly reduced. We conclude that mucin secretion by cultures of human tracheobronchial gland cells is independent of Cl(-) secretion, at baseline, and is unaltered in CF; that the ratio of Cl(-) secretion to mucus secretion varies markedly depending on mediator; and that secretions induced by stimulation of β-adrenergic receptors will be abnormally concentrated in CF.     

Role of cyclic AMP-dependent protein kinase activation in regulating rat submandibular mucin secretion

The extent of activation of rat submandibular gland cyclic AMP-dependent protein kinase (EC 2.7.1.37) was determined in vitro using dispersed cells to assess the involvement of this enzyme in submandibular mucin secretion. cAMP-dependent protein kinase activation, as determined by activity ratio method, was markedly increased following β-adrenergic receptor activation. 0.5 M NaCl was required in the homogenization buffer for stabilization of the hormonally activated cAMP-dependent protein kinase. A role for cAMP-dependent protein kinase activation in regulating mucin secretion was strongly suggested by the following: (1) the kinase activity ratio increased rapidly after β-adrenergic receptor stimulation; (2) dose-response relationship of the kinase activation following β-adrenergic receptor activation correlated with isoproterenol induced mucin release; (3) termination of β-adrenergic mediated mucin secretion caused a rapid decrease in the kinase activity ratio; (4) dibutyryl cyclic AMP stimulation caused an increase in the kinase ratio; whereas (5) pure cholinergic and pure α-adrenergic receptor stimulation had no effect on endogenous kinase activity. Although cAMP-dependent protein kinase activation may not be the only regulator of mucin secretion, these data suggest an important regulatory role for this kinase activation during rat submandibular mucin release.     

E-cadherin promotes EGFR-mediated cell differentiation and MUC5AC mucin expression in cultured human airway epithelial cells

In previous work, we showed that epidermal growth factor receptor (EGFR) activation causes mucin expression in airway epithelium in vivo and in human NCI-H292 airway epithelial cells and normal human bronchial epithelial (NHBE) cells in vitro. Here we show that the cell surface adhesion molecule, E-cadherin, promotes EGFR-mediated mucin production in NCI-H292 cells in a cell density- and cell cycle-dependent fashion. The addition of the EGFR ligand, transforming growth factor (TGF)-alpha, increased MUC5AC protein expression markedly in dense, but not in sparse, cultures. MUC5AC-positive cells in dense cultures contained 2 N DNA content and did not incorporate bromodeoxyuridine, suggesting that they develop via cell differentiation and that a surface molecule involved in cell-cell contact is important for EGFR-mediated mucin production. In support of this hypothesis, in dense cultures of NCI-H292 cells and in NHBE cells at air-liquid interface, blockade of E-cadherin-mediated cell-cell contacts decreased EGFR-dependent mucin production. E-cadherin blockade also increased EGFR-dependent cell proliferation and TGF-alpha-induced EGFR tyrosine phosphorylation in dense cultures of NCI-H292 cells, suggesting that E-cadherin promotes EGFR-dependent mucin production and inhibits EGFR-dependent cell proliferation via modulation of EGFR phosphotyrosine levels. Furthermore, in dense cultures, E-cadherin blockade decreased the rate of EGFR tyrosine dephosphorylation, implicating an E-cadherin-dependent protein tyrosine phosphatase in EGFR dephosphorylation. Thus E-cadherin promotes EGFR-mediated cell differentiation and MUC5AC production, and our results suggest that this occurs via a pathway involving protein tyrosine phosphatase-dependent EGFR dephosphorylation.