Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Mucus, which covers the airway luminal surface, is essential in protecting the epithelial surface from environmental insults. The major components in the secretion that contribute to the viscoelastic properties of mucus derive from the large molecular weight of mucous glycoproteins (mucins). There are at least 12 human mucin genes (MUCs), and eight of these (MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, and MUC8) are expressed, at least at the message level, in airway epithelium (1). For these eight MUC messages, translation products of MUC1, MUC2, MUC5AC, and MUC5B have been found in the airway epithelium, but only MUC5AC and MUC5B products have been convincingly demonstrated to be major components in airway mucus secretion (4, 5). The regulation of the secretion and synthesis of mucin in airway is poorly understood. Accordingly, mucin secretion, depending on the mediators, can be regulated through nitric oxide-dependent pathways (6) or receptor G-protein-dependent signaling (7). Compared with mucin secretion regulation, the pathway in the regulation of MUC expression is still quite meager. Recently, it was demonstrated that mitogen-activated protein kinase (MAPK)/extracellular regulated kinase (MEK) 1 and 2 were involved in the transcriptional activation of MUC5AC and/or MUC2 by bacteria (8), air pollutant residual oil fly ash (ROFA) (9), and tobacco smoke (10). Airway mucin is secreted in vivo mainly by surface goblet and submucosal gland cells. Mucus-cell metaplasia and hypermucus secretions are clinical symptoms that are frequently associated with various human airway diseases, such as asthma, chronic bronchitis, and cystic fibrosis (CF) (11).

Extracellular nucleotide triphosphates (NTPs), such as adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP), exert potent physiologic actions on a variety of tissues and cell types (14). In the airways, both ATP and UTP activate Cl secretion in both CF and non-CF airway epithelia (15, 16). In addition, both ATP and UTP are equally potent in regulating goblet cell-mediated mucin release (17). In parallel studies with alveoli, both ATP and UTP were equally potent in the stimulation of surfactant release (18). The equipotency and equi-effectiveness of ATP and UTP in regulating these activities has suggested that the P2Y₂ receptor (formerly P2U) is predominantly responsible for these activities. P2Y receptors are first classified pharmacologically, on the basis of the potency order of agonists (19). Several members of this receptor family have been either cloned or identified, including five cloned mammalian receptorsP2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁and two uncloned putative receptors, P2Y_ADP (or P_2T) receptor and endogenous uridine nucleotide-specific receptors (pharmacologically similar to cloned P2Y₄ and P2Y₆ receptors). All of these are G-protein-coupled receptors (GPCRs) (19). It has been reported that membrane phospholipases such as phosphatidylinositol phospholipase (PL)C (7), PLA₂ (20), phosphatidylcholine (PC)- PLC, and PC-PLD (21) are coupled to ATP receptors via G-protein in various cell types.

For this study, we examined the effects of extracellular NTP on airway mucin production. To our surprise, UTP is the only nucleotide examined that is capable of stimulating mucin (MUC5AC and MUC5B) expression as well as mucin secretion in human airway epithelial cells. This in vitro finding could be extended to an in vivo mouse airway by an intratracheal instillation of UTP-saline. This is the first demonstration in an airway system that UTP acts differently from ATP on the regulation of airway mucin gene expression and secretion. Preliminary studies with various inhibitors demonstrated that separate signaling pathways are involved in UTP regulation of mucin secretion and MUC expression. These results suggest a significant role for UTP in regulating airway mucous-cell differentiation.

	Materials and Methods

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Cell Culture and Inhibitor Treatment

Human tracheobronchial tissues were obtained from the Medical Center of the University of California at Davis (Davis, CA), Sacramento Organ Donation Foundation, Inc. (Sacramento, CA) and the Anatomic Gift Foundation (Laurel, MD), with consent. The University of California at Davis Human Subjects Review Committee approved all procedures involved in tissue procurement. We obtained 10 tracheobronchial tissues from various donors for this study. These tissues included two from patients with no obvious pulmonary disease at the time of death, four with pulmonary fibrosis, three with chronic obstructive pulmonary disease, and one with emphysema. We successfully established primary cultures from these tissues. Tracheobronchial epithelial (TBE) cell isolation and culture methods were carried out as described previously, with some modifications (22). Primary human TBE cells (1 or 2 × 10⁴ cells/cm²) were plated on a collagen gel substratum-coated Transwell chamber (25 mm) in Ham's F12/Dulbecco's modified Eagle's medium (DMEM) (1:1) supplemented with insulin (5 µg/ml), transferrin (5 µg/ml), epidermal growth factor (EGF) (10 ng/ml), dexamethasone (0.1 µM), cholera toxin (10 ng/ml), bovine hypothalamus extract (15 µg/ml), and bovine serum albumin (0.5 mg/ml). All-trans-retinoic acid at 0.3 µM was added immediately after plating. Preparation of these hormonal supplements has been described elsewhere (22). Under retinoid influence, human TBE cells underwent mucous-cell differentiation in culture after confluence. These primary TBE cultures, after 1 wk in immersed cultured condition, were shifted to an air- liquid interface culture condition. Under the biphasic culture condition, further mucociliary differentiation of human TBE cells was observed. After 3 wk in culture (2 wk under the biphasic culture condition), primary human TBE cells underwent mucociliary differentiation, including cilia and mucus-secreting granules formation (22). It was under such a well-differentiated stage that various extracellular NTPs were added. Cultures were terminated 2 h to 4 d later for various measurements of mucin secretion and RNA analysis.

Initial inhibition experiments all used published inhibitor concentrations on epithelial cells from literature. To further determine the optimal dose for those inhibitors, we also checked concentrations up to 5-fold above or below those published concentrations. In the present report, all concentrations mentioned are optimal concentrations based on the literature and our experiments. For all the inhibitor experiments (except pertussis toxin), the cells were incubated in the Ham's F12/DMEM (1:1) with 0.3 µM all-trans-retinoic acid for 24 h, and preincubated with inhibitors for 2 h. Afterwards, cells were exposed to UTP plus inhibitors, to UTP only, or to inhibitors only for 2 h at mucin secretion measurement and 16 h for RNA harvest. For pertussis toxin and its B-oligomer treatment, cells were incubated in the Ham's F12/DMEM (1:1) with 0.3 µM all-trans-retinoic acid and 100 ng/ml pertussis toxin (or B-oligomer) for 24 h, and all the following steps were similar to those in the other inhibitor treatment.

Mucin Secretion and Enzyme-Linked Immunosorbent Assay

Monoclonal antibodies (17Q2) were used to measure mucin secretion from cultured cells (22). Previous studies have shown that mucin secretion occurs on the apical side of the biphasic culture condition (22), and so only the medium from the apical surface was collected for this study to determine the level of mucin secretion. Before the drug treatment, the apical surface of the biphasic TBE culture was washed twice with fresh culture medium (1 ml per wash per chamber) to remove various deposits. This was followed by a 2-h incubation with 0.5 ml fresh culture medium on the apical surface. After preincubation, conditioned medium (CM) on the apical surface was collected and combined with a wash of 0.5 ml fresh medium, which was designated as a basal secretion (b-CM). Fresh medium containing various amounts of NTPs was added onto both the apical surface (0.5 ml) and the lower chamber area (2 ml). After 2 h incubation in a 37°C CO₂-incubator, the CM in the apical surface was collected and combined with the wash (0.5 ml). This second combined medium was designated as the treatment secretion (CM-NTP). The levels of mucin in these b-CM and CM-NTP secretions were quantified by a double-sandwich enzyme-linked immunosorbent assay (ELISA) method, as described previously (23). Purified sputum mucin from a pool of patients was used as a standard and the quantity was expressed as nanograms of protein per milliliter, which was determined by amino-acid compositional analysis (23). The sensitivity of the ELISA was 0.5 to 16 ng/ml. Most of the primary human TBE cells cultured under the described culture conditions produced 30 to 80 ng of mucin/d/million cells. For determining the effect of NTP on mucin secretion, the relative mucin secretion rate between the treated CM-NTP and b-CM-NTP was compared with that in the control without the drug treatment (CM-C and b-CM-C) during these two periods, in the same set of primary cultures. The relative stimulatory or inhibitory effects on mucin secretion by a drug treatment was expressed as percent of (CM-NTP/b-CM-NTP)/(CM-C/b-CM-C) × 100%. Each assay involved triplicate Transwell chambers, and experiments were repeated independently from three primary cultures derived from different donors, with similar results.

RNA Isolation and Northern Blot Hybridization

At Day 21 after plating, RNA was isolated from cultures by single-step phenol-chloroform extraction (24). For Northern blot hybridization, equal amounts of total RNA (20 µg/lane) were subjected to electrophoresis on a 1.0% agarose gel in the presence of 2.2 mM formaldehyde and transblotted onto Nytran membranes. The RNA was crosslinked to membrane by a UV Stratalinker 2400 (Stratagene, La Jolla, CA). Membrane prehybridization and hybridization with ³²P-labeled MUC5B- or MUC5AC-specific oligonucleotide probes were carried out as described elsewhere (24). Single-stranded antisense oligonucleotides corresponding to the tandem repeat unit of human MUC5B and MUC5AC, 5'-TGT GGT CAG CTT TGT GAG GAT CCA GGT CGT CCC CGG AGT GGA GGA GGG-3' (423nt-376nt; Genbank: U63836) and 5'-AGG GGC AGA AGT TGT GCT GGT TGT GGG AGC AGA GGT TGT GCT GGT TGT-3' (582nt-535nt; Genbank: Z34277), respectively, were end-labeled with -³²P-ATP by T4 polynucleotide kinase. All the blots described in the present report were exposed overnight to the phosphor screen (Molecular Dynamics, Sunnyvale, CA) and read using the STORM system (Molecular Dynamics). The relative abundance of MUC5B/MUC5AC message in Northern blot was normalized with 18S ribosomal RNA band.

Intratracheal Instillation of NTP-Saline Solutions on Mouse Airway

Female BALB/C mice (8 wk old) were purchased from Charles River Laboratories, Inc. (Wilmington, MA) These mice were anesthetized by Avertin (2.5% solution, 0.15 ml/10 g intraperitoneally). The upper portion of trachea was surgically exposed by making an incision in the skin and separating the thyroid gland and overlying muscles and connecting tissues. A microsyringe carrying a 27-gauge needle filled with NTP-saline solutions was used for injection into the exposed trachea. About 50 µl of solution was injected into the lumen of each mouse trachea. After injection, the skin was sutured and the animals were recovered to resume their daily activity. The control animals were injected with saline solution only. At Days 1, 2, and 3 after injection, mouse trachea and lung were removed and fixed in a formalin solution, and then processed for paraffin blocks preparation. Paraffin sections were prepared and processed for both Alcian blue (AB)- periodic acid-Schiff (PAS) staining and in situ hybridization.

In Situ Hybridization

Single-stranded antisense oligonucleotides corresponding to the tandem repeat unit of mouse muc5ac (25) and 3' end sequence of mouse muc5b (Chen and colleagues, manuscript in preparation) were synthesized. These sequences were: 5'-GGT TGT AGA GAT GGT GCT GGT CTT TCC TGT ATT GGG TGA GCT GGT TTG-3' (102nt-55nt; Genbank: L42292) and 5'-GCA GGA ACC CTC GCA GAA GGT GAT GTT GAC CTC TGT CTC ACA GCC CTT-3', respectively. These DNAs (100 pmol each) were end-labeled with the Dig oligonucleotide tailing kit (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer's protocol. Sense oligonucleotides corresponding to these sequences were also synthesized and used as a control for the hybridization. In situ hybridization was carried out on the basis of the manufacturer's instructions. Briefly, slides were digested with 10 µg/ml Proteinase K in 50 mM Tris-Cl (pH 8.0) and 50 mM ethylenediaminetetraacetic acid for 15 min at 37°C, rinsed twice in 0.2× saline sodium citrate (SSC), and then postfixed in 4% paraformaldehyde/phosphate-buffered saline for 20 min. Slides were treated twice for 5 min each time with 0.1 M triethanolamine (TEA) (pH 8.0) and blocked by 0.25% acetic anhydride in 0.1 M TEA. The sections were then dehydrated through the ethanol series. For each section, 0.5 pmol digoxigenin-labeled oligonucleotide probe in 50 µl of hybridization buffer was applied. The hybridization buffer contained 2× SSC, 1× Denhardt's solution, 10% dextran sulfate, 50 mM phosphate buffer (pH 7.0), 50 mM dithiothreitol, 250 µg/ml yeast transfer RNA, 100 µg/ml poly A, and 500 µg/ml salmon-sperm DNA. The section was hybridized at 45°C overnight in a humidified chamber. After the hybridization the section was washed twice for 15 min each time at 37°C with 2× SSC, twice for 15 min each time with 1× SSC, and twice for 15 min each time with 0.25× SSC. After the washes, the slide was reacted with anti-Dig primary antibody conjugated with the alkaline phosphatase. After several washes, the reacted probes in the slide were color-developed with the Dig Nucleic Acid Detection kit (Roche Molecular Biochemicals).

Reverse Transcriptase/Polymerase Chain Reaction Characterization of P2Y Receptors

Three UTP-responsive P2Y receptors have been described (19). To verify the existence of these receptors in primary cultures of human TBE cells, a semiquantitative reverse transcriptase/polymerase chain reaction (RT-PCR) approach was carried out. Briefly, primers corresponding to each receptor were synthesized. For P2Y₂ receptor, primers were: forward, 5'-GCA GAT CCG ACA GAA CTG AC-3'; reverse, 5'-GCA AAC TCA GCC CTC ATT AC-3'. P2Y₆, primers were: forward, 5'-TTC CTG CCT TTT CAC ATC ACC-3'; reverse, 5'-GTC TGT CCA TCT CCA TGC CC-3'. For P2Y₄, they were: forward, 5'-ACA ACA GCA ACA AAG GGA C-3'; reverse, 5'-CTA TCC TCA GGG CAG GGA CAC-3'. For -actin, primers were: forward, 5'-TCA CCC ACA CTG TGC CCA TCT ACG A-3'; reverse, 5'-CAG CGG AAC CGC TCA TTG CCA ATG G-3'. PCR DNA products from these primers were verified by DNA sequencing.

Statistical Analysis

Data are expressed as means ± standard deviation on the basis of triplicates in each experiment and at least two independent experiments from two different primary tissues. Group differences were analyzed by analysis of variance. When P < 0.05, the difference was considered significant.

Materials

The sources of chemicals used were as follows: adenosine(A), adenosine 5'-diphosphate (ADP), ATP, ATP--S, ,-methylene ATP (AMPCPP), and UTP from Sigma Chemical Co. (St. Louis, MO); and uridine 5'-diphosphate (UDP), pertussis toxin, pertussis toxin B-oligomer, U73122, U73343, PD98059, and Calphostin C from Roche Molecular Biochemicals.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Stimulation of Mucin Secretion in Primary Cultures of Human TBE Cells by NTPs

At Day 21, human TBE cells grown under an air-liquid interface culture condition were exposed to various NTPs and their analogues (0.1 mM). As shown in Figure 1, the relative mucin secretion rate was near 75%, having been elevated by UTP and ATP. However, the stimulation was reduced when nucleotide diphosphates UDP and ADP were used. Without phosphate, a purine base, A, had no stimulatory effect. The notion that NTP, rather than the metabolites, is directly involved in the regulation was further supported by treatment with ATP--S, which is a nonhydrolyzable analogue. For another ATP analogue, AMPCPP, the stimulation was low but higher than A. We also carried out a dose study, and the results (data not shown) were consistent with previous reports (26). Overall, we observed a potency order of UTP  ATP  ATP--S > UDP  ADP > AMPCPP >> A in the stimulation of mucin secretion. This result is consistent with a previous report using primary cultures of hamster and human airway epithelial explants (26). On the basis of the equal potency of ATP and UTP, it has been suggested that a P2Y₂ receptor is involved in the regulation of mucin secretion.

View larger version (33K): [in this window] [in a new window]   Figure 1.   Effects of nucleotides on mucin secretion in human TBE cultures. TBE cells were cultured as described in MATERIALS AND METHODS and treated with various nucleotides (0.1 mM), as indicated. Secreted CM was collected from each culture chamber as described and the mucin content in the secreted medium was quantified by a double-sandwich mucin ELISA method, as described elsewhere (23). The relative stimulatory or inhibitory effects on mucin secretion by these nucleotides were determined as described in MATERIALS AND METHODS.

Upregulation of MUC Expression (MUC5AC, MUC5B) by UTP but Not by ATP and Other Analogues

Initially, we examined the effects of various nucleotides and NTPs at various doses (0 to 1 mM) on the regulation of MUC expression. As shown in Figure 2, UTP was the only one in the group, except UDP, that could stimulate MUC5B expression in culture. UDP at the 1-mM level had much less stimulatory effect. These experiments were repeated in all of the primary TBE cultures derived from these 10 human tissues. Results were the same as shown in Figure 2. A time-course study was then carried out to determine the duration of UTP treatment required to elevate MUC expression. As shown in Figure 3, UTP at 10⁴ M could elevate MUC5B and MUC5AC messages in a time-dependent manner. Significant elevation of MUC5B and MUC5AC messages was observed 6 and 12 h, respectively, after UTP treatment, and the elevation continued over time. At 96 h after the treatment, there was a 5- to 10-fold elevation of MUC5B and MUC5AC messages in culture. For MUC5AC, the message was less abundant than it was for MUC5B in culture. Thus, the stimulations at the early time points were not as obvious as those seen for MUC5B. These results were repeated in three of the primary cultures.

View larger version (79K): [in this window] [in a new window]   Figure 2.   Effects of nucleotides and nucleotide triphosphates on MUC5B expression in human TBE cultures. Experiments were carried out as described in Figure 1. Cultures were treated with these nucleotides and nucleotide triphosphates overnight at 1 mM. RNAs were isolated and used for Northern blot hybridization.

View larger version (62K): [in this window] [in a new window]   Figure 3.   Time-course study of effects of UTP on MUC5AC and MUC5B expression in human TBE cultures. Experiments were carried out as described in Figure 2. RNAs were isolated at various times after UTP (0.1 mM) treatment.

This elevation was quite specific for UTP. We found that UTP stimulated both MUC5B and MUC5AC message levels in a dose-dependent manner (Figure 4). For both messages, the significant increase was seen with 0.01 mM UTP. For other nucleotide analoguesATP, ADP, A, ATP--S, and AMPCPPeven at the 1-mM level, there was no stimulatory effect (Figure 2). However, UDP had a slightly stimulatory effect on the MUC5B message (Figure 2). These dose-dependent studies were repeated in three of the primary TBE cultures. Results were very similar to Figure 4.

View larger version (77K): [in this window] [in a new window]   Figure 4.   Effects of UTP doses on MUC5AC and MUC5B expression in human TBE cultures. Experiments were carried out as described in Figure 2. RNA samples were collected from cultures after UTP treatment for 6 (MUC5B) and 24 h (MUC5AC), respectively.

Elevation of Mucous Cell Population in Mouse Airways by an Intratracheal Instillation of UTP

To extend the in vitro study described earlier to in vivo, an intratracheal instillation of UTP- and ATP-saline solutions was carried out on pathogen-free mice. We observed an elevation of AB-PAS positive-stained cell population in mouse airway epithelia, reflecting an increase of mucous-cell population. As shown in Figure 5, 48 h after a single instillation of 0.1 mM UTP (50 µl/mouse), AB-PAS- positive-stained cells were readily seen in mouse trachea (Figure 5, A5 and A6) and in the distal airway regions (Figure 5, B5 and B6), as compared with saline-treated controls, which showed no positive-stained cells (Figure 5, A1, A2, B1, and B2). For an intratracheal instillation of 0.1 mM ATP, the majority of tracheal and lung sections showed no AB-PAS- positive cells except in one or two areas, as shown in Figure 5, A3, A4, B3, and B4. To confirm that these positively stained cells in UTP-treated mouse airways are indeed mucous cell-like, in situ hybridization was carried out. As shown in Figure 6, most of these positively stained cells expressed both mouse muc5b and muc5ac messages.

View larger version (83K): [in this window] [in a new window]   Figure 5.   Effects of UTP/ATP-saline intratracheal instillation on mouse airways. UTP/ATP salines were instilled intratracheally on a mouse as described in MATERIALS AND METHODS. At 48 h later, mouse trachea and lung tissues were fixed and processed for paraffin section and AB-PAS staining. (A) Sections of mouse upper airway regions. A1, A2: Saline control instillation; A3, A4: ATP (0.1 mM, 50 µl/ mouse) instillation; A5, A6: UTP (0.1 mM, 50 µl/mouse) instillation. A1, A3, and A5 are at ×10 original magnification; A2, A4, and A6 are enlarged pictures (original magnification, ×60) corresponding to the marked regions in A1, A3, and A5, respectively (marked by empty boxes). (B) Sections of mouse lung. B1, B2: Saline control instillation; B3, B4: ATP (0.1 mM, 50 µl/mouse) instillation; B5, B6: UTP (0.1 mM, 50 µl/mouse) instillation. B1, B3, and B5 are at ×10 original magnification; B2, B4, and B6 are enlarged pictures (original magnification, ×60) corresponding to the marked regions in B1, B3, and B5, respectively (marked by empty boxes).

View larger version (115K): [in this window] [in a new window]   Figure 6.   In situ hybridization of mouse muc5ac and muc5b message on mouse airway tissue sections after UTP instillation. Experiments were carried out as described in Figure 5. In situ hybridization was carried out as described in MATERIALS AND METHODS. This figure combines three pictures from serial sections of the same tissue block. The background picture is from the AB-PAS- staining picture. The enlarged pictures show positive hybridization by using mouse muc5ac and muc5b probes separately on two other sections.

Demonstration of the Presence of UTP-Selective Receptors in Human Primary TBE Cells

Because UTP is so potent in increasing mucin secretion and elevating MUC expression, we hypothesized that those effects were mediated by UTP-selective receptors. Currently, three cloned P2Y receptorsP2Y₂, P2Y₄, and P2Y₆ have a high response to UTP. Using semiquantitative RT-PCR, we could detect all three receptors in primary human TBE cells (Figure 7). Sequencing of these PCR bands confirmed the nature of these P2Y receptors. Among them, P2Y₂ and P2Y₆ receptor messages were abundant, whereas the P2Y₄ receptor level was low.

View larger version (43K): [in this window] [in a new window]   Figure 7.   RT-PCR amplification of P2Y subtype receptors (P2Y2, P2Y4, and P2Y6). The amplified PCR products of -actin act as control. RNA collection and experiment conditions were carried out as described in MATERIALS AND METHODS.

Inhibitor Studies of Signaling Pathways Involved in UTP-Enhanced Mucin Secretion and MUC5B Expression

To determine whether the same signaling pathway involved in ATP/UTP-dependent mucin secretion is also involved in UTP-enhanced MUC expression, various inhibitors used in the studies from other laboratories (7, 26) were tried. Because MUC5AC message was low in the culture, we focused on MUC5B expression. The first task was to determine whether the stimulation could be inhibited by pertussis toxin (100 ng/ml), which could ADP-ribosylate members of G_i/o family G-proteins, uncoupling them from their cognate receptors and thereby inhibiting the signaling through such receptors (7). As shown in Figures 8 and 9, UTP-enhanced mucin secretion and MUC5B expression, respectively, could be blocked by the inhibitor, suggesting a G protein-dependent pathway for both enhancements. For further validation of this inhibitory effect, pertussis toxin B-oligomer (100 ng/ml) was used to treat the cells. B-oligomer is the pentameric cell-binding component of pertussis toxin, which is responsible for facilitating the entry of the enzymatic subunit into receptive cells. As shown in Figures 8 and 9, B-oligomer showed no effects on mucin secretion and MUC5B expression. Thus, the inhibitory effects elicited by pertussis toxin must reside on the enzymatic component which entered into receptive cells. For PLC and protein kinase (PK) C inhibitors, U73122 (10 µM) and Calphostin C (0.1 µM), respectively, the reactions to these two inhibitors were quite different among these UTP-dependent effects. These chemicals inhibited the stimulation of UTP-dependent mucin secretion from the 75 to the 25% level (Figure 8). In contrast, these two inhibitors had no effect on UTP-enhanced MUC5B message level (Figure 9). Higher doses of these inhibitors had some cytotoxic effects on cells but UTP-dependent MUC5B expression was not affected (data not shown). U73343, an analog of U73122 and a very weak PLC inhibitor, was also used to verify the effect of U73122. As shown in Figures 8 and 9, U73343 had no significant effects. Because it was proposed that the MAPK pathway might be involved in the regulation of MUC5AC and MUC2 expression (8), we decided to test whether the UTP-dependent MUC expression could be inhibited by PD98059 (a MEK inhibitor), commonly used for the inhibition of the MAPK pathway. As shown in Figure 9, PD98059, having a slight enhancement of basal MUC5B message level, could block UTP-dependent stimulation of MUC5B expression. However, it had no inhibitory effect on UTP-dependent stimulation of mucin secretion even at the 75-µM level (Figure 8).

View larger version (35K): [in this window] [in a new window]   Figure 8.   Effects of inhibitors on UTP-dependent stimulation of mucin secretion in human TBE cultures. Experiments were carried out as described in MATERIALS AND METHODS and in Figure 1. Data are indicated as percentage increase compared with the control. C, UTP-treated only; Pert, UTP and pertussis toxin- treated; PD, UTP and PD98059-treated. U73122, UTP and U73122-treated; Cal, UTP and Calphostin C-treated; U73343, UTP and U73343-treated; PertB, treated with UTP and pertussis toxin B-oligomer.

View larger version (53K): [in this window] [in a new window]   Figure 9.   Effects of inhibitors on UTP-dependent stimulation of MUC5B expression in human TBE cultures. Inhibitor treatments were as described in MATERIALS AND METHODS, and experiments were carried out as described in Figure 2. Labels in the figure as follows: C, control; UTP, UTP-treated only; Pert, pertussis toxin- treated only; PertB, pertussis toxin B-oligomer-treated only; Pert+UTP, pertussis toxin and UTP-treated; PertB+UTP, treated with pertussis toxin B-oligomer and UTP; U73122, U73122-treated only; U73343, U73343-treated only; U73122+UTP, U73122 and UTP-treated; U73343+UTP, U73343 and UTP-treated; Cal, Calphostin C-treated only; Cal+UTP, treated with Calphostin C and UTP; PD, PD98059-treated only; PD+UTP, PD98059 and UTP-treated.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the present study we demonstrate a unique dual role played by UTP in the regulation of mucin secretion and gene expression in human TBE cells. Until now no other nucleotides examined have those dual capabilities in increasing both mucin secretion and MUC expression. The significance of this in vitro finding is strengthened by the in vivo study of the mouse model in which the airway mucous-cell population was elevated dramatically after a single intratracheal instillation of UTP-saline. ATP and its analogues had stimulatory effects on mucin secretion; however, they did not stimulate MUC expression. An intratracheal instillation of ATP solution had no significant effect on mouse airway mucous-cell population. This is the first demonstration that UTP acts differently from ATP in the regulation of airway mucous-cell differentiation.

The second significant finding in this study is the result of different signaling pathways exerted by a single agent, UTP, on the regulation of mucin secretion and MUC expression. These pathways are apparently initiated at the receptor-G-protein complex, inasmuch as both UTP-enhanced phenomena were inhibited by pertussis toxin, which has been known to interrupt a signal transduction system by ADP-ribosylating some members of G_i/0 family G-proteins. However, after this step the signaling that regulates these two events varies. For UTP- and ATP-dependent stimulation of mucin secretion, the pathway is PLC/ PKC-dependent. Our results are consistent with a previous report on hamster cells (7, 28), which notes the potency of various nucleotide analogues and sensitivity to various inhibitors. However, we provide additional information that the MAPK pathway is not involved in the secretion regulation. This is based on the use of the MEK inhibitor PD98059. This information cannot be explained by the lack of sensitivity of human TBE cells to this inhibitor because a similar treatment in the same primary culture was able to block UTP-dependent MUC expression. In contrast to the secretion, the regulation of MUC expression by UTP is independent of PLC/PKC pathways but affected by the MAPK system. This is based on the finding that the stimulation is not inhibited by both PLC and PKC inhibitors, but it is inhibited by the MEK inhibitor.

How can UTP act on two different pathways? The simplest explanation is that there are two GPCRs involved. One regulates mucin secretion, the other regulates MUC expression. Accordingly, the former GPCR belongs to the P2Y₂ subtype on the basis of the order of potency for various nucleotides: UTP  ATP  ATP--S > UDP  ADP > AMPCPP >> A. Kim and colleagues (7) have suggested in a hamster surface epithelial cell system that upon the binding of UTP or ATP, PLC was activated with a release of diacylglycerol, which subsequently set off the PKC activity. This pathway fits well with the current inhibitor study. For the latter GPCR the pathway is still unclear, perhaps in part because this is the first demonstration that UTP has a unique effect on MUC expression and there is insufficient information available to determine the purinergic receptor subtype. However, on the basis of the potency of effects between pyrimidine- and purine-based nucleotides, the receptor subtype for UTP-induced MUC expression is close to the P2Y₄ and P2Y₆ subtypes. The semiquantitative RT-PCR data support the presence of these two subtypes in primary human TBE cells. Cressman and colleagues have recently theorized that a dinucleotide pyrimidine receptor, P2Y₆, is perhaps mediating UDP-stimulated airway epithelial Cl secretory response (29). Kim and colleagues have also demonstrated that UTP is not a good competitor for ATP--S³⁵-specific receptor in hamster airway surface epithelial cells (27). These studies support the theory of a pyrimidine-specific receptor in airway epithelial cells.

However, the pharmacologic responses and expression profiles of the two cloned pyrimidine-responsive receptors (P2Y₄ and P2Y₆) do not completely fit with our results. For the P2Y₄ subtype, it has been reported that P2Y₄ is UTP-selective (19), which is quite consistent with our demonstration of the unique role of UTP. However, the expression of P2Y₄ is very restrictive, with a low expression level in the lung (19). Our RT-PCR results also reflect this. The P2Y₆ subtype, even though it expresses in a wide tissue range, has generally been accepted to be a UDP-selective receptor with higher responsiveness to UDP than to UTP (19). However, in the present study, we found that UDP has some stimulatory effect on MUC expression but much less than that of UTP. Perhaps the observed UTP effects are the new pharmacologic manifestation of those P2 receptors generated by the interaction between the subtypes and their specific cellular environment. It is also possible that a new pyrimidine-responsive receptor subtype which has not been identified before is responsible for this UTP-enhanced MUC expression. Further study is needed to elucidate such a subtype.

We also found that the downstream signaling responsible for UTP-dependent MUC expression is through the MAPK pathway. This finding is consistent with Basbaum and colleagues' reports in which they have shown the involvement of the MAPK pathway in the transcriptional activation of MUC5AC and/or MUC2 expression by bacterial products, by the air pollutant ROFA, or by tobacco smoke (10). We do not have nuclear run-on and messenger RNA stability analyses for this UTP effect. It has been suggested before that MUC messages are quite stable in cells (30). However, post-transcriptional regulation has been implicated in elastase-induced MUC5AC message levels (31). It is possible that both transcriptional and post-transcriptional regulations are involved in this UTP-dependent MUC expression.

There are several ways for GPCRs to initiate MAPK activation. One of the major pathways involves the activation of PLC and subsequently PKC (7, 28). Because PLC and PKC inhibitors have no inhibitory effects on UTP-specific activity, it is unlikely that this pathway plays any significant role in UTP-enhanced MUC expression. The second large group of mediators linking GPCR with MAPK is the receptor or nonreceptor tyrosine kinase, which includes EGF (32, 33), platelet-derived growth factor (34), Src (35, 36), etc. Upon autophosphorylation or phosphorylation of membrane-bound substrates, tyrosine kinase can create docking sites where proteins containing phosphotyrosine-binding domains (PTB and SH2) can assemble in multimeric-signaling complexes leading to activation of Ras, and subsequently to the activation of the MAPK pathway. And Src/Ras/MAPK pathway has been demonstrated to play an essential role in transcriptional activation of MUC5AC by the supernatant of Gram-negative bacteria (8). The current analysis of signaling molecules used by GPCRs to stimulate MAPK in a variety of cellular systems shows there are additional routes that may cooperate with, or even substitute for, those tyrosine kinase-based pathways, including protein tyrosine phosphatase SH-PTP1 (37), Ras-guanine-nucleotide releasing factor (38), kinase suppressor of Ras-1 (39) and PI3K (40). Therefore, further study will be needed to find the signaling mediators between the UTP-specific GPCR and the activation of MAPK pathway in the UTP-dependent MUC expression.

In summary, the present study demonstrates that UTP is the only known nucleotide that can both induce mucin secretion and elevate MUC expression through different signaling pathways. This phenomenon can be repeated in mice by an intratracheal instillation of UTP. On the basis of the short-term Cl secretion-stimulating effects of UTP, one of the UTP derivatives has recently been used in a clinical trial in treating CF by restoring chloride transport and increasing the lung clearance in a patient with CF (16). But our data shows that UTP treatment could increase MUC expression and subsequently increase mucous-cell population, and suggests that long-term medication using UTP-related drugs may have serious side effects.