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Jul 6, 2007 - secretory pathway and the quantity of mucin stores in goblet cells, respectively. ... release from goblet cells might be stimulated (see Figure 1), ...
Page 1 of 39 Articles in PresS. Am J Physiol Lung Cell Mol Physiol (July 6, 2007). doi:10.1152/ajplung.00150.2007

Regulation of Airway Goblet Cell Mucin Secretion by Tyrosine Phosphorylation Signaling Pathways Lubna H. Abdullah1 and C. William Davis1,2,3 1

Cystic Fibrosis/Pulmonary Research and Treatment Center, and 2 Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, NC 27599

Running head: Tyrosine phosphorylation signaling and mucin secretion

Correspondence: C. William Davis, PhD Cystic Fibrosis/Pulmonary Research and Treatment Center 6009 Thurston-Bowles University of North Carolina, Chapel Hill, NC 27599 USA 919-966-7060 (voice) 919-966-7524 (FAX) [email protected]

Copyright © 2007 by the American Physiological Society.

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Abstract Mucus hyperproduction in pulmonary obstructive diseases results from increased goblet cell numbers, and possibly increased cellular mucin synthesis, occurring in response to inflammatory mediators acting via receptor tyrosine kinases (RYK) and tyrosine phosphorylation (Y-Pi) signaling pathways. Yet, increased mucin synthesis does not lead necessarily to increased secretion, as mucins are stored in secretory granules and secreted in response to extracellular signals -- commonly assumed to be mediated by G-protein-coupled receptors (GPCRs). We asked whether activation, [i] of Y-Pi signaling pathways, in principal, and [ii] of nPKCdelta by Y-Pi, specifically, might lead to regulated mucin secretion. nPKCdelta in SPOC1 cells was tyrosine phosphorylated by exposure to purinergic agonist (ATPgammaS) or PMA, actions that were blocked by the Src kinase inhibitor, PP1. Mucin secretion, however, was not affected by PP1. Hence, activation of nPKCdelta by Y-Pi is unlikely to participate in GPCR-related mucin secretion. Mucin secretion from both SPOC1 and normal human bronchial epithelial (NHBE) cells was stimulated by generalized protein Y-Pi induced by the tyrosine phosphatase inhibitor, pervanadate. Pervanadate-induced SPOC1 cell mucin secretion was not affected by inhibition of Src kinases (genistein or PP1), or of PI3 kinase (LY-294002). MAP kinase pathway inhibitors, RAF1 Kinase Inhibitor-I and U-0126 (MEK), inhibited SPOC1 cell pervanadate-induced secretion by ~50%. Significantly, the phospholipase C inhibitor, U73122, essentially abolished pervanadate- and ATPgammaS-induced mucin secretion from both SPOC1 and NHBE cells. Hence, phospholipase C signaling may play a key role in regulated mucin secretion, whether the event is initiated by mediators interacting with GPCRs or RYKs.

Keywords: lung, mucus, mucins, exocytosis

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Introduction Mucus hyperproduction in the airways is characteristic of all the pulmonary obstructive diseases (e.g., chronic bronchitis/COPD, cystic fibrosis, asthma, primary ciliary dyskinesia). Its fundamental cause is thought to be an inflammation-driven proliferation of goblet cells in the superficial epithelium and of mucous cells in submucosal glands. In those diseases best understood, cystic fibrosis and extrinsic asthma, the expansion of mucin producing cells appears to result, respectively, from the hyperabsorption of liquid from airway surfaces and attendant bacterial infections (8), and from exposure to environmental allergens and/or toxins (27; 29). Recent work suggests that a variety of tyrosine receptor kinase (RYK) based pathways mediate the effects of pathogenic and environmental insults in the airways to stimulate hyperplasia and/or metaplasia of mucin-secreting cells and the upregulation of mucin gene expression, or less specifically, increased PAS-positive staining in the airways (e.g., 5; 14; 15; 44). Increased mucin gene expression and PAS staining, however, define only the very beginning of the mucin secretory pathway and the quantity of mucin stores in goblet cells, respectively. We require substantially more knowledge to understand fully the complex relationships between mucin gene transcription and translation, the mechanisms and regulation of the extensive post-translational modifications of mucins and their packaging into vesicles and granules, and finally, the exocytic release of mucins from constitutive-like and regulated secretory pathways. Agonist regulated mucin secretion has received a fair amount of attention in recent years (18; 19; 32; 60); however, most of this work has focused on G-protein coupled receptor (GPCR) signaling pathways, under control conditions, rather than on factors that initiate exocytosis in an inflammatory, mucin hypersecretory environment. In the studies reported here we examined potential intracellular tyrosine phosphorylation (Y-Pi) signaling pathways by which mucin release from goblet cells might be stimulated (see Figure 1), following two general approaches. Page 2

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First, recent work has shown that mucin secretion from cultured SPOC1 (1; 2; 46) and several human and non-human airway epithelial cell culture models (16; 18; 31; 32; 60) is regulated intracellularly by diacylglycerol (DAG)- and Ca2+- based pathways, with ATP and UTP, acting via apical membrane P2Y2 receptors, representing the predominant agonists. An important action of diacylglycerol in cells is the activation of conventional and novel isoforms of protein kinase C (see 38), and in a wide range of goblet cell models activation of PKC by the phorbol ester, PMA, has very consistently resulted in a stimulation of mucin secretion (18; 19; 31; 32). In SPOC1 cells, cPKC , nPKC , , and , and aPKC and / are expressed, but only cPKC and nPKC respond to PMA by translocating in the classic fashion, from cytosol to membrane fractions (1). Furthermore, only nPKC so responded to stimulation by purinergic agonist: its translocation into the membrane fraction was dependent on the agonist concentration and correlated well with the degree of mucin secretion. More recently, however, we found (manuscript submitted) that overexpression of nPKC in SPOC1 cells had no effect on the mucin secretory response, whereas cells overexpressing nPKC exhibited an enhanced response. Similarly, mucin secretion was unaffected in isolated, perfused agonist-exposed tracheas of nPKC deficient mice, whereas the response was nearly abolished in tracheas of nPKC mice. In sum, these results suggest nPKC as the active PKC isoform in agonist-regulated mucin secretion; however, since the nPKC isoform can be activated by Y-Pi (see 55), in addition to DAG/PMA and the usual cofactors (38), there is the strong possibility that this isoform is responsive to the effects of inflammatory mediators to trigger mucin release. We therefore tested whether activation of nPKC by Y-Pi induces regulated mucin secretion. Second, most of the intracellular regulatory pathways activated by inflammatory factors and environmental insults possess component proteins which are activated by Y-Pi. For

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instance, mucin gene expression can be driven by bacterial pathogens interacting with RYKs (35; 51), mucous meta- and hyperplasia in the airways is driven in part by RYKs activated by IL-4, IL-13, and other cytokines (15; 50; 54), and the effects of neutrophil elastase to stimulate mucin gene expression are likely mediated by Y-Pi signaling pathways (40; 48; 52) . In this paper we found that a general increase in tyrosine phosphorylation of cellular proteins stimulated mucin secretion, and then we used a variety of well-characterized inhibitors in an attempt to define the signaling pathway(s) responsible for observed stimulation.

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Materials and Methods Materials. Culture medium was purchased from GibcoBRL (Gaithersburg, MD) and its supplements from Collaborative Research (Bedford, MA). Nucleotides were purchased from Boehringer-Mannheim (Indianapolis, IN). PKC isoform-specific, polyclonal antibodies generated against synthetic peptides corresponding to unique C-terminal sequences were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), as was a monoclonal phosphotyrosine antibody (p-Tyr). The inhibitor compounds LY-294002, D-609, and U-73122 were purchased from Biomol (Plymouth Meeting, PA), and U0126, wortmannin, and RAF1 Kinase Inibitor-I were from Calbiochem (San Diego, CA). Unless specified otherwise, all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Pervanadate (PV) was prepared immediately before use by combining 100 µM Na vandadate with 100 µM H2O2, in DMEM/F12 (57).

SPOC1 Cell Culture and mucin assay. SPOC1 cells (3; 42), passage 7 – 14, were grown as described previously (1), in 100 mm tissue culture plates or in 6-well cluster plates (Costar, Cambridge, MA). Except for cells grown solely for passaging, the medium contained 10 nM retinoic acid. Culture media were changed daily, and the cultures were used for experiments after differentiation of a mucin secreting phenotype, 17 - 20 days post-confluence. Media samples were assessed for mucin content by enzyme-linked lectin assay (ELLA) as described previously (3), using 100 µl samples bound to 96-well high-binding microtiter plates (Costar #3590 ) during an overnight-4 oC, or a 2 hour-37 oC, incubation, followed by a 1 hr incubation with SBA lectin. After stopping the reaction with 4 M sulfuric acid, the optical density was determined at 490 nm (Dynatech model MR5000 microtiter plate reader; Chantilly, VA), and

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optical density was converted to ng mucin from standard curves using purified SPOC1 mucin applied to each microtiter plate (3). Normal human bronchial epithelial cell culture and mucin assay. Human bronchial epithelial (NHBE) cells were obtained from normal human bronchi, in accordance with Institutional Review Board-approved protocols, and grown in primary culture as described previously (23; 45). Briefly, NHBE cells were isolated and grown on plastic culture dishes in bronchial epithelial cell growth medium (BEGM), passaged at ~80% confluence, and firstpassage cells were seeded onto collagen-coated, 12-mm Transwell-Clear permeable supports (TCols; Costar) at 250,000 cells per support. After confluence, the cells were maintained under air-liquid interface (ALI) conditions in ALI culture medium (BEGM modified per Ref 23), which was changed at the basolateral surface three times a week. NHBE cell cultures were used for experiments 4–6 wk after confluence, when the columnar cells are well differentiated as ciliated or goblet cells. NHBE cells used for mucin secretion experiments were carefully washed using the protocol of Kemp, et al. (31), and incubated 30 min without or with secretagogue. Mucins secreted onto the culture luminal surfaces were sampled by gentle 400 µl collection in DMEM/F12 and quantified by ELISA using a ‘subunit’ antibody which recognizes most vertebrate polymeric mucins as described previously (manuscript submitted), following the general ELLA protocol above but using purified NHBE mucins as a standard (28). Western blotting/immunoprecipitation/tyrosine phosphorylation. Proteins phosphorylated on tyrosine residues were identified from whole SPOC1 cell lysates using a p-Tyr antibody. Whole cell lysates prepared and protein concentrations estimated as previously described (2) were frozen and stored at –80 oC. Within 48 hours of the experiment, samples were thawed, equivalent amounts of protein (10 – 20 µg/lane) were resolved by 10% SDS/PAGE and electrophoretically transferred to nitrocellulose membranes, and the blots probed with p-Tyr Page 6

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antibody. In studies examining the Y-Pi of nPKC , specifically, the isoform was first immunoprecipitated from whole cell lysates using a nPKC antibody. nPKC antibody (1 µg) was added to cell lysates and incubated for 2 hrs, then Protein A agarose beads (20 µl, Santa Cruz Biotechnology) were added and incubated overnight (~16 hr), both at 4 oC with gentle shaking. The beads were collected by micro-centrifugation, 15 min at 3,000 RPM. After 3X wash with ice cold PBS, the beads were collected, resuspended in 2X LSB, the proteins resolved by SDS-PAGE (20 µg protein/lane), transferred to nitrocellulose membranes, and the blots probed with the p-Tyr antibody. Immunoreactive proteins in all blots were detected using enhanced chemiluminescence (Amersham Corporation, Arlington Heights, IL). Films of the Western blots were digitized to 12 bits with a platform scanner at a resolution of 1200 x 1200 dpi (UMAX Powerbook 1000), and quantified using Metamorph image processing software (Universal Imaging, Downington, PA).

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Results Tyrosine phosphorylation of nPKC and mucin secretion. In principle, activation of nPKC by Y-Pi may occur at the membrane following nPKC translocation in response to DAG/PMA or agonist (6; 34), or it may occur in the cytosol independent of translocation (39). When activated, nPKC is free to phosphorylate target proteins in sub-plasma membrane or cytosolic compartments, until it is deactivated by dephosphorylation. We tested whether nPKC was tyrosine phosphorylated following stimulation of SPOC1 cells with purinergic agonist or PMA, and determined whether this event correlated with stimulated mucin secretion. Figure 2A shows that the state of nPKC Y-Pi changes rapidly following stimulation by agonist and PMA. In response to the application and continuous presence of ATP S1, nPKC was tyrosine phosphorylated transiently, with its peak activation occurring at 1 min. By 10 min following application, the amount of nPKC Y-Pi had declined approximately to control levels, and these levels continued to decline with longer periods of incubation until they were ~25 % below control levels at 60 min. NPKC

was also activated by Y-Pi following stimulation by 30 or 300 nM PMA. The

lower, 30 nM concentration of PMA induces a maximal translocation response from nPKC and cPKC in SPOC1 cells, but mucin secretion is stimulated maximally at 300 nM (1; 46). The two concentrations of PMA induced a Y-Pi of nPKC that was essentially maximal at 10 min, with the higher concentration causing a higher level of phosphorylation, ~3-fold (300 nM) above control vs. ~2-fold (30 nM). In contrast to activation by ATP S, nPKC Y-Pi following high PMA exposures was relatively stable: the staining intensities on Western blots at the 60 min time points were similar to or greater than those at the 5 min time point. The effects of lower PMA concentra73122tions declined slightly from the peak. Page 8

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Isoproterenol is a specific agonist for -adrenergic receptors and has been shown to stimulate cAMP production in SPOC1 cells (11), but neither SPOC1 cells nor NHBE cells response to cAMP-dependent signals with increases in mucin secretion (2; 16). In contrast to both ATP S and PMA, isoproterenol caused an inhibition of nPKC Y-Pi, to levels ~25 % below control, with a time course that was both rapid and persistent. As will be appreciated from the blot in Figure 2A, the isoproterenol data were rather noisy relative to those derived from the other secretagogues. The reasons for this difference in quality are not clear, but on average the data indicated that nPKC Y-Pi was diminished following isoproterenol. The effects on mucin secretion from SPOC1 cells was assessed in the same preparations used for the experiments on the time course of nPKC Y-Pi (Figure 2B). Consistent with our previous results (1), ATP S and 30 nM PMA stimulated mucin secretion from these cells to approximately the same degree, and 300 nM PMA had greater effects. Note that the mucin secretory response to ATP S was significantly increased at 1 min, relative to controls, whereas the responses to the two levels of PMA was not different from control at the initial time point. Thereafter, the cells responded to 30 nM PMA in a similar manner to ATP S, with mucin secretion reaching a plateau at >10 min, whereas 300 nM PMA elicited a response that increased throughout the duration of the experiment. As expected, isoproterenol had no significant effect on mucin secretion from these cells. Comparing Figures 2A and 2B reveals that PMA-induced mucin secretion appeared to follow the pattern of nPKC phosphorylation, i.e., both nPKC Y-Pi and mucin secretion had relatively stable plateaus.

For ATP S, in contrast, both secretion and nPKC Y-Pi were

elevated quickly (1 min) but then diverged, with nPKC Y-Pi declining thereafter while mucin secretion rose to a plateau. Isoproterenol caused a rapid inhibition of nPKC Y-Pi, but had no

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effect on mucin secretion. These data suggest that activation of nPKC by Y-Pi in SPOC1 cells may not correlate temporally with sustained mucin secretion (i.e., > 1 min). Two tyrosine kinase inhibitors were tested for effects on nPKC Y-Pi elicited by ATP S and PMA, genistein, a non-selective tyrosine kinase inhibitor, and PP1, an inhibitor of Src family kinases (24) that also inhibits some other tyrosine kinases (56). As shown in Figure 3A, genistein had no effect on nPKC Y-Pi by any of the activators tested, whereas PP1 dramatically reduced nPKC -Y-Pi induced by both ATP S and PMA. Neither of these compounds, however, had a significant effect on agonist- or PMA-stimulated mucin secretion from SPOC1 cells (Figure 3B), suggesting the absence of a relationship between activation of nPKC by tyrosine phosphorylation and regulated mucin secretion.

Generalized tyrosine phosphorylation of cellular proteins. Since there did not appear to be a specific relationship between nPKC Y-Pi and mucin secretion, we tested whether a generalized protein Y-Pi in SPOC1 cells stimulated mucin secretion. For this purpose, we used the tyrosine phosphatase inhibitor, pervanadate (49). As shown in Figure 4A, treatment of SPOC1 cells with PV promoted the Y-Pi of numerous proteins in whole cell extracts with time courses that varied significantly, but none of which developed in a major way within a 1 min time frame. Generalized protein Y-Pi was increased to an obvious degree at 15 min, and by 30 min it was massive. PP1 had obvious inhibitory effects on the Y-Pi of some proteins at 1 min and on many proteins at the 15 min time point, and over longer times genistein was also effective at reducing the degree of phosphorylation (Figure 4A, inset).

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PV also elicited mucin secretion from SPOC1 cells, and it enhanced secretagogue induced secretion (Figure 4B). PV stimulated resting cells above their baseline mucin secretion rates by 5.6 ± 0.7 fold, and this degree of stimulation, at least in this experiment, was similar to the effects of purinergic agonist and 30 nM PMA in the absence of PV. When cells were exposed to a combination of PV and secretagogue, the effects were additive. Notably, neither genistein nor PP1 had any significant effect on measured baseline, PV-, secretagogue-, or PV plus secretagogue-stimulated mucin release from SPOC1 cells.

Effects of other tyrosine phosphorylation pathway inhibitors on pervanadate-induced mucin secretion. To test whether the effects of PV could be ascribed to specific effects on cellular signaling pathways other than Src kinases, we challenged each of 4 ATP S- or PVexposed SPOC1 cell passages to a series of widely used inhibitors, targeting a variety of key intracellular signaling pathways (see Figure 1). In the experimental matrix, each inhibitor was tested at three concentrations, in half or full log increments ranging upward from a lowest concentration near the IC50 of each compound (the data in parentheses below are for the highest concentrations tested). In these experiments (Figure 5), PV stimulated mucin secretion, as in the experiment for Figure 4, above. Note, however, that the absolute magnitude of the responses to agonist and PV were both somewhat reduced, by about 50%, compared to the data of Figure 4. For the Figure 4 experiments, ATP S and PV stimulated relative increases in mucin secretion of 7.3 ± 0.9 and 5.6 ± 0.7 fold , whereas for Figure 5 the changes were 11.3 ± 2.6 and 3.8 ± 0.5 fold, respectively. The difference in relative PV-induce mucin secretory responses between the two experiments (Figures 4 and 5) could be due to variable pervanadate concentrations generated during its preparation, to variations in PV-induced protein Y-Pi of SPOC1 cells caused by tyrosine phosphatase inhibition, variable effects of Y-Pi on signaling pathways, or numerous Page 11

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other possibilities. Notably, in all experiments, both those presented herein as well as all our preliminary experiments, PV was as consistent in causing a significant release of mucins from SPOC1 cells release as are P2Y2 purinergic agonists, i.e., it was only the magnitude of the PVinduce secretory responses that was variable. The most profound inhibitory effect on PV-induced SPOC1 cell mucin secretion was observed with U73122, a specific inhibitor of phospholipase C (PLC; 7; Figure 5): this compound abolished the mucin secretory response of SPOC1 cells to ATP S (R% = 102±7 @30 µM), and it was nearly as effective against PV-induced mucin secretion (R% = 88±18 @30 µM). The so-called PC-specific PLC inhibitor, D-609, had no measurable effect on either secretory response (data not shown). Wortmannin, an inhibitor of PIP3 production by PI3 kinase (4; 59), also had substantial inhibitory effects on the SPOC1 cell mucin secretory response to ATP S and to PV. The wortmannin inhibition of the PV response (R% = 87±2 @ 10µM) was significantly greater than its effect on ATP S induced mucin secretion (R% = 57±15, 100 µM, p < 0.05). LY294002, however, another key PI3 kinase inhibitor (4; 59), had no effect on SPOC1 cells exposed to either ATP S (R% = 4±1 @ 30 µM) or PV (R% = 0±8 @ 30 µM). Because the mucin secretory response was not inhibited by both PI3 kinase-active compounds, and because we had to use concentrations of wortmannin well above its known nM IC50 against PI3 kinase to observe any inhibiton of mucin secretion, the observed effects of wortmannin are likely due to its well known inhibition of myosin light chain kinase (4; 37) and/or PI4 kinases (4; 17; 59). We also tested the effects of two inhibitors of the MAP kinase pathway (Figure 1), RAF1 Kinase Inhibitor-I (33), and the MEK inhibitor, U-0126 (20). Neither of these inhibitors had significant effects on the ATP S-induced SPOC1 cell mucin secretory response. Both

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compounds, however, had moderate and highly variable, but concentration-dependent inhibitory effects on PV-induced secretion (RAF1 Kinase Inhibitor-I, R% = 53 ± 36 @ 10 µM; U-0126, R% = 49 ± 26 @ 10 µM). Hence, these studies indicate that PLC and the MAP kinase pathways may mediate the effects of inflammatory stimuli acting on Y-Pi signaling pathways to stimulate regulated mucin secretion, with PLC likely representing the primary signaling pathway.

Effects of pervanadate on normal human bronchial epithelial cell cultures. As with any study using a cell line as its primary material, it is important to determine whether the observations have biological and clinical relevance. To this end we compared the effectiveness of purinergic agonist and PV in stimulating mucin secretion from primary cultures of NHBE cells. As shown in Figure 6, both ATP S and PV stimulated mucin release from the cultures. Similar to the SPOC1 cell experiment in Figure 5, the approximately 2-fold increase in mucin secretion from NHBE cell cultures exposed to PV was significant, but less than the mucins released by ATP S (1.91 ± 0.1 versus 3.5 ± 0.5 fold (p < 0.05, n = 10, respectively). Notably, the PLC inhibitor, U73122, abolished the effects of both ATP S and PV to induce mucin secretion from NHBE cultures (R% = 101.6 ± 13.5 and 110.3 ± 24.6, respectively). Hence, PV stimulates mucin release from human goblet cells via a PLC-sensitive pathway, similar to SPOC1 cells.

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Discussion The regulation of airway goblet cell mucin secretion has been fairly well detailed over the past two decades. In general terms, regulated mucin secretion is very similar to the regulated exocytic pathways of the endocrine secretory cells and the neuronal synaptic machinery (9; 10; 12; 58), with an extracellular agonist initiating a cellular messenger cascade which results in the exocytic release of secretory cargo. For airway goblet cells, the primary agonist appears to be luminal ATP or UTP interacting with apical membrane P2Y2 purinoceptors (18; 19; 32), which couple with PLC to generate IP3 and DAG, mobilizing Ca2+ and activating PKC, respectively (see 60). Substantial evidence supports this scenario: [i] treating intact cells with ionomycin (2; 16; 31) or elevating bulk Ca2+ in the solution bathing cells permeabilized with Streptolysin-O (46) stimulates mucin release, as does [ii] treating cells with the DAG mimic, PMA (18; 19; 31; 32); [iii] measured intracellular Ca2+ in agonist-stimulated cells increases following a time course consistent with mucin release (45); [iv] treating cells with the PLC inhibitor, U73122, blocks both Ca2+ mobilization (45) and mucin release (Figures 5 and 6); and [v] stimulating tracheas of nPKC deficient mice with agonist results in a greatly reduced mucin secretory response (manuscript submitted). This view of regulated mucin secretion, however, was developed using a classical approach of cell physiology which may not be relevant to inflammatory environments. Hence, our findings that nPKC is activated in correlation with mucin secretion (1), whereas it is nPKC , not nPKC that is essential for a full agonist-induced mucin secretory response (manuscript submitted), were provocative for suggesting the possibility that nPKC could be activated by tyrosine phosphorylation and mediate the goblet cell mucin secretory response to inflammatory insults (40; 48; 21;21; 53). Implicit in this suggestion is the notion that regulation of mucin gene transcription and glycoprotein synthesis are independent of Page 14

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the regulated exocytic release of mucin granules; however, this is not to say that certain inflammatory pathways might not stimulate both activities (see Figure 1), especially given the known Y-Pi signaling pathways operative in inflammatory cell secretion (see 43).

Activation of NPKC by tyrosine phosphorylation. nPKC is a novel, Ca2+-independent isoform that is unique in several respects from other PKC isoforms. A primary difference is its activation by Y-Pi, in addition to the DAG/PMA mode of activation typical of the conventional and novel PKC subfamilies (38; 41; 55). Notably, activation of nPKC by Y-Pi can take place at the plasma membrane by Src family kinases as part of GPCR or RYK mediated cell stimuli (6; 34), or in the cytosol (39), and it endows the isoform with the ability to operate independently of the plasma membrane (see 55). nPKC has been suggested to be involved in mucin secretion from airway goblet cells activated by both purinergic agonist (1) and neutrophil elastase (40), agents which most likely operate via GPCR and RYK pathways, respectively (18; 32; 40; 48; 52). We first showed that nPKC is tyrosine phosphorylated in SPOC1 cells as part of purinergic agonist and PMA stimulation (Figure 2). The onset of nPKC Y-Pi in response to purinergic agonist was rapid, but transient, whereas with PMA it was slower to develop, but persistent. The mucin secretory responses to these secretagogues also differed, developing more rapidly with purinergic agonist. Significantly, the Src family kinase inhibitor, PP1, abolished nPKC Y-Pi stimulated by both purinergic agonist and PMA, whereas it had no effect on the mucin secretory responses elicited by these secretagogues (Figure 3). Similarly, PP1, and genistein, had no effect on the mucin secretory response caused by generalized PV-induced protein Y-Pi (Figure 4). Hence, Src family kinases are unlikely to participate in the regulation of mucin secretion, whether they are

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activated by GPCRs, RYKs, or other pathways. Moreover, the results indicate that nPKC is also unlikely to participate in the mucin regulatory response when activated by Y-Pi. This latter conclusion is strengthened by our recent observations in SPOC1 cells overexpressing PKC isoforms and gene-targeted mice deficient for individual PKCs. In this study, nPKC , not nPKC was essential for a full, purinergic agonist-induced tracheal mucin secretory response (manuscript submitted). Together, these studies suggest strongly that nPKC is not involved in regulated mucin secretion. The data indicating activation of nPKC during the goblet cell response to purinergic agonist (1; manuscript submitted), neutrophil elastase (40; 48), or to inflammatory mediators (21; 53), therefore more likely relate to goblet cell mucin gene transcription (13; 22; 26; 47; 62), mucin glycoprotein synthesis, or other mucin secretory pathway activities downstream from exocytic release.

Generalized protein Y-Pi and mucin secretion. In determining whether signaling pathways involving Y-Pi other than those involving nPKC were capable of stimulating mucin secretion from SPOC1 cells, we wished to avoid the use of known inflammatory mediators, such as TNFU (21), for the uncertainties in their underlying cellular signaling paths. Rather, we used PV to stimulate a generalized Y-Pi of cellular proteins, taking advantage of its known effect to inhibit tyrosine phosphatase activity (49). Not surprisingly, PV caused a massive increase in tyrosine phosphorylated proteins in SPOC1 cells, and it also stimulated mucin secretion of the same order of magnitude as secretagogue-induced responses (Figures 4 and 5). Moreover, PV also caused a similar release of mucins from NHBE cells (Figure 6). Notably, the experiments determined the mucins released over a 30 min, a period of time too brief for significant mucin glycoprotein synthesis to complicate the secretion results.

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Having achieved a mucin secretory response to cellular Y-Pi, we next tested a variety of common inhibitors to test whether specific signaling pathways might be implicated in the response (see Figure 1). In these experiments, we always compared the effects of inhibitors on PV- and purinergic agonist-induced mucin secretion. Somewhat unexpectedly, several inhibitors with a rather wide spectrum of inhibitory effects in the aggregate had no effect on either PV or agonist-induced SPOC1 cell mucin secretion, namely genistein, PP1, and LY-294002 (Figures 3 and 4, and see Results). These negative results indicated the likely lack of involvement of Src family kinases and PI 3 kinases in agonist or Y-Pi signaling pathways leading to goblet cells mucin secretion. Two inhibitors of the MAP kinase signaling pathway, RAF1 Kinase Inhibitor-I, and the MEK inhibitor, U-0126, selectively inhibited PV-induced SPOC1 cell mucin secretion by ~ 50%. That the MAP kinase inhibitors had no effect against agonist-induced secretion suggests that the inhibition of PV-induced secretion is specific, and that inflammatory insults which activate associated RYKs may stimulate both goblet cell mucin biosynthesis and secretion, at least to some degree. The most effective degree of inhibition of PV-induced SPOC1 cells mucin secretion, ~90%, was observed with the PLC-specific inhibitor, U73122. Interestingly, U73122 inhibited agonist-induced secretion even more strongly, i.e., it was totally abolished (Figure 5), and it also inhibited both PV- and agonist-induced secretion from NHBE cells (Figure 6). That PLC should be involved in agonist-induced goblet cell mucin secretion is not surprising, since P2Y2 purinoceptors are known to couple to and activate PLC, via Gq, with the subsequent release of IP3 and DAG driving the cellular pathways leading to mucin granule exocytosis (see Introduction). The notion that PLC might also mediate Y-Pi signaling pathways leading to regulated mucin secretion, however, is relatively novel, although PLC-V has been long known to participate in RYK signal transduction (see 30). Many recent studies have examined the role of Page 17

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RYKs and/or Y-Pi pathways in the regulation of airway mucin production, with a focus on mucin gene expression (for review: 5; 14; 35; 36; 61). Studies of this type, however, consistently neglect consideration of the signals necessary to initiate release of the newly synthesized mucins. Hence, the present finding is important for the parsimonious conclusion that regulated mucin secretion is initiated by inflammatory mediators acting via RYKs activating PLC, as do GPCR-mediated signals. PLC, of course, is not a single enzyme, but a family of some 13 isoforms organized in 6 different subfamilies. The four isoforms of PLC-W isoforms typically couple to GPCRs via GUq and GWV, and the two PLC-V isoforms are activated by RYKs (see 25; 30). The multiplicity of PLC isoforms, however, strengthens, rather than diminishes the central finding of this study. It emphasizes how a signaling cascade may be initiated at different sites by different signals, then proceed down a common pathway of effector molecules to ultimately stimulate mucin granule exocytosis.

In conclusion, we find that generalized protein tyrosine phosphorylation stimulates mucin secretion from SPOC1 and NHBE goblet cells, suggesting that inflammatory mediators operating via Y-Pi signaling pathways, in principal, may stimulate mucin release in addition to other likely effects on the mucin secretion pathway. PLC represents the likely primary pathway mediating the effects of these agents, though the MAP kinase pathway may also be involved to some degree. Activation of nPKC by tyrosine phosphorylation, however, appears not to relate directly to regulated mucin secretion, even though it appears to be activated in a correlative manner to mucin secretion. Most likely, nPKC is involved at points in the mucin secretory pathway upstream of the regulated exocytic release of mucins, possibly with mucin gene transcription.

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Acknowledgements The authors received financial support for these studies in the form of grants from the National Heart, Lung, and Blood Institute (HL063756) and the North American Cystic Fibrosis Foundation.

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Footnotes 1

ATP S is an analog of ATP that serves as a full agonist in whole cell responses of SPOC1 cells

(3). It is used instead of ATP or UTP in these studies because it is resistant to hydrolysis by ecto-nucleotide hydrolyzing enzymes, and because it activates P2Y2 over other purinoceptors (60).

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Figure Legends [for reviewer convenience, the figure legends are duplicated below each figure] Figure 1. Cellular signaling pathways. This highly schematized and simplified scheme of cellular signaling shows GPCR (purple) and RYK (blue) pathways that potentially affect mucin secretion. Note that the Jak/STAT and TRADD/NFKB pathways affect gene expression selectively, so would not be expected to affect mucin secretion directly or acutely. The cellular messengers known to regulate exocytosis are indicated in white on a green background. Proteins that are boxed and indicated in red were targeted by the inhibitors indicated in parentheses, to test for potential effects on mucin secretion.

Figure 2. Time course of nPKC tyrosine phosphorylation and of mucin secretion in SPOC1 cells following stimulation. After washing, SPOC1 cultures were incubated for 30 min with or without ATP S (purinergic agonist, 100 µM), PMA (30 or 300 nM), or isoproterenol (W-agonist, 10 µM). At the indicated time points, culture media were removed from cultures for mucin assays, and the cells were lysed. nPKC immunoprecipates (IP) from the cell extracts were resolved by PAGE (20 µg protein/lane) and assessed by Western blotting.

A. nPKC Y-

Pi, top panel shows a sample nPKC IP blot probed with a p-Tyr antibody. Bottom panel shows the integrated intensities of the immunoblots normalized to the degree of staining at t = 0. Data are expressed as the mean ± SE fold-change from control (n = 8-10) . Note that bars descending from 1.0 indicate a decline in phosphorylation, relative to control. B. Mucin secretion. At each time point for nPKC -Y-Pi determinations above, the medium was assessed by ELLA for mucin content.

For convenience, the t=0 time points are not shown

note that the t=1 min points for

all but agonist were not different from their respective controls. * = p