Regulation of human airway mucins by acrolein and inflammatory mediators

Michael T. Borchers, Michael P. Carty, and George D. Leikauf

Department of Environmental Health, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0056


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Bronchitis, asthma, and cystic fibrosis, marked by inflammation and mucus hypersecretion, can be caused or exacerbated by airway pathogens or irritants including acrolein, an aldehyde present in tobacco smoke. To determine whether acrolein and inflammatory mediators alter mucin gene expression, steady-state mRNA levels of two airway mucins, MUC5AC and MUC5B, were measured (by RT-PCR) in human lung carcinoma cells (NCI-H292). MUC5AC mRNA levels increased after >= 0.01 nM acrolein, 10 µM prostaglandin E2 or 15-hydroxyeicosatetraenoic acid, 1.0 nM tumor necrosis factor-alpha (TNF-alpha ), or 10 nM phorbol 12-myristate 13-acetate (a protein kinase C activator). In contrast, MUC5B mRNA levels, although easily detected, were unaffected by these agonists, suggesting that irritants and associated inflammatory mediators increase mucin biosynthesis by inducing MUC5AC message levels, whereas MUC5B is constitutively expressed. When transcription was inhibited, TNF-alpha exposure increased MUC5AC message half-life compared with control level, suggesting that transcript stabilization is a major mechanism controlling increased MUC5AC message levels. Together, these findings imply that irritants like acrolein can directly and indirectly (via inflammatory mediators) increase airway mucin transcripts in epithelial cells.

aldehyde; chronic obstructive pulmonary disease; cytokine; eicosanoids


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

EXCESSIVE MUCUS SECRETION is a hallmark in the pathogenesis of several airway diseases including chronic bronchitis, asthma, and cystic fibrosis (34). Patients suffering from these diseases have pathological abnormalities in both the submucosal glands and surface epithelium, characterized by inflammation, increased mucous cell number, and excessive airway mucus. Several classes of inflammatory mediators have been implicated in the process of mucus hypersecretion based on their ability to stimulate secretion from cultured cells and tissue explants. These include cytokines [e.g., tumor necrosis factor-alpha (TNF-alpha )] (12, 31), proteases (27, 50), reactive oxygen species (2), and arachadonic acid metabolites (17, 36). The mechanisms controlling mucus secretion are not completely understood but are likely to involve protein kinase (PK) C- and calcium-dependent signaling pathways (1).

Mucus glycoproteins (mucins) provide airway secretions with their characteristic adhesiveness, elasticity, and viscosity. Mucins are large heterogeneous proteins (20,000-30,000 kDa) that vary in length from 200 to 4,000 nm (49). As much as 80% of their molecular mass consists of carbohydrate side chains linked by glycosyltransferases to serine and threonine residues of the peptide backbone (52). Mucin peptides are encoded by multiple genes characterized by DNA sequences with variable numbers of tandem repeat domains. These sequences vary in composition and size (>50% of protein) but have the common feature of being rich nucleotide sequences encoding threonine, serine, and proline (18). Nine distinct mucin genes (MUC1-MUC4, MUC5AC, MUC5B, and MUC6-MUC8) have been described in the respiratory, gastrointestinal, and reproductive tracts (reviewed in Ref. 47). Of these, MUC5AC and MUC5B proteins have been isolated from human airway secretions and are considered to be major constituents of the mucous layer (40, 45, 48, 53). The large size and complex repetitive nature of mucin genes have hindered the study of cellular events involved in mucus synthesis and secretion. Control of mucin synthesis likely involves both transcriptional (32, 35) and posttranscriptional mechanisms (56) that regulate mRNA formation and stability, protein translation and glycosylation, and granule formation and exocytosis.

Mucus hypersecretion can also be experimentally induced in response to irritants including tobacco smoke (46). Acrolein (CH2==CHCHO) is a potent irritant aldehyde present in high concentrations [50-70 parts/million (ppm)] in tobacco smoke (5) and is a constituent of wood smoke, diesel exhaust, and photochemical smog (3). Previously, Borchers et al. (7) reported that rats exposed to acrolein develop increased numbers of epithelial mucous cells containing MUC5AC and that mucus hypersecretion in the airways was preceded by increases in steady-state MUC5AC mRNA levels. Exposure to acrolein also results in acute and chronic airway inflammation (30, 33) and increased release of the arachadonic acid metabolites prostaglandin E2 (PGE2) and 15-hydroxyeicosatetraenoic acid (15-HETE) (16, 30). Because these mediators are known agonists of mucus secretion (17, 36), their formation after acrolein exposure represents a possible mechanism by which mucin synthesis is controlled.

The present study was designed to determine whether mucin genes were regulated at the mRNA level by irritant exposure and inflammatory mediators. Toward this goal, we investigated the effects of acrolein, eicosanoids, TNF-alpha , and a PKC activator on MUC5AC and MUC5B steady-state mRNA levels in human lung carcinoma cells. Because steady-state mRNA levels are controlled by transcription rate and mRNA stability, we also examined the ability of TNF-alpha to increase message half-life.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental design. To determine whether acrolein, inflammatory mediators, or a PKC agonist affects mucin mRNA expression, cultured cells derived from a human lung cancer (NCI-H292) (10) were exposed to acrolein, PGE2, 15-HETE, TNF-alpha , phorbol 12-myristate 13-acetate (PMA), or vehicle control for 4 h (37°C, pH 7.4). To determine whether the effects on mucin gene expression were unique to NCI-H292 cells, another human lung carcinoma cell line (A549) (19) was exposed to TNF-alpha or PMA. NCI-H292 and A549 cells were selected because increased MUC5AC mRNA levels are accompanied by increased MUC5AC protein synthesis and mucus secretion in these cells (26, 58). After exposure, the cells were lysed, total RNA was isolated, and mucin (MUC5AC and MUC5B) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were analyzed by RT-PCR. In preliminary studies with NCI-H292 cells, maximal MUC5AC induction by each agonist occurred between 1 and 4 h. Agonist concentrations were selected based on previous studies (17, 31, 36) that demonstrated that similar doses stimulate mucus secretion in epithelial cell explants and cultures. To examine whether TNF-alpha regulates MUC5AC mRNA levels by posttranscriptional mechanisms, NCI-H292 cells were treated with TNF-alpha or vehicle control for 2 h, followed by the addition of 5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole (DRB), an RNA synthesis inhibitor (59).

Cell culture. NCI-H292 (a human pulmonary mucoepidermoid carcinoma) and A549 cells (human lung carcinoma) were purchased from the American Type Culture Collection. The cells were grown in 75-cm2 plastic tissue culture flasks (Costar, Cambridge, MA). NCI-H292 or A549 cells were maintained in RPMI 1640 medium or Dulbecco's modified Eagle's medium (DMEM; GIBCO BRL, Life Technologies, Grand Island, NY), respectively, each supplemented with 10% fetal calf serum (BioCell, Rancho Dominguez, CA), penicillin (100 U/ml), and streptomycin (100 µg/ml; both from Sigma, St. Louis, MO) (37°C, pH 7.4). The cells were seeded at a density of 5,000 cells/cm2 and passaged at ~90% confluence.

Acrolein and agonist exposures. NCI-H292 or A549 cells were seeded (5,000 cells/cm2) into six-well plates (Corning) and grown to confluence. The cells were incubated (37°C, pH 7.4) for 20 h in serum-free medium (RPMI 1640 or DMEM) and subsequently treated with each agent in phosphate-buffered saline (PBS; GIBCO BRL). The cells were treated for 4 h (37°C, pH 7.4) with acrolein (0.01-100 nM; Aldrich, Milwaukee, WI), 15-HETE (10 µM; Sigma), PGE2 (10 µM; Sigma), TNF-alpha (1 nM; R&D Systems, Minneapolis, MN), or vehicle (0.05% ethanol) in PBS. After exposure, the medium was removed, and the cells were lysed in 4 M guanidine thiocyanate (Fisher, Fair Lawn, NJ). The resulting solution was stored at -70°C until RNA isolation.

MUC5AC mRNA stability. MUC5AC message half-life and the effect of TNF-alpha treatment on mRNA stability was examined by exposing NCI-H292 cells to the pharmacological transcription inhibitor DRB (59). In these experiments, cells were treated for 2 h with TNF-alpha (1 nM) to achieve maximum MUC5AC levels. At time 0, DRB (100 nM) was added, and the cells were incubated for an additional 0, 1, 2, 4, 8, or 16 h. After each exposure period, the cells were lysed, total RNA was isolated, and MUC5AC mRNA levels were determined by RT-PCR analysis. Degradation of c-myc mRNA (half-life approx  15 min) was also examined by RT-PCR to evaluate DRB activity.

RNA isolation and RT-PCR analysis. Total RNA was isolated from cultured cells by the guanidinium-phenol-chloroform procedure described by Chomczynski (11). The purity was estimated by spectrophotometric determination of the 260- to 280-nm absorption ratio. Purified RNA was stored at -70°C until analyzed by RT-PCR. RT-PCR was chosen for these experiments because smeared signals (continuous bands of multiple sizes) were produced by Northern analysis of mucin mRNAs, resulting in difficulties in quantification (55, 57). In contrast, a single band was generated by RT-PCR by selecting primers in the nonrepeat regions of the mucin genes.

For RT-PCR, primers were generated from published sequences of MUC5AC (38), MUC5B (15), GAPDH (54), and c-myc (13). All primers except c-myc (Clontech, Palo Alto, CA) were synthesized by the University of Cincinnati (OH) DNA Core Facility. Primer sequences were as follows: MUC5AC lower primer (position 1945), 5'-ACT TGG GCA CTG GTG CTG-3'; MUC5AC upper primer (position 1283), 5'-TCC GGC CTC ATC TTC TCC-3' (product length 680 bp); MUC5B lower primer (position 2688), 5'-CAG TGG CAG AGG CCG TGC AGT A-3'; MUC5B upper primer (position 106), 5'-CAG GGC ATT TGG ACA GTT TTT C-3' (product length 544 bp); GAPDH lower primer, 5'-TGC TGG GGC TGG TGG TC-3'; GAPDH upper primer, 5'-TCA AGT GGG GCG ATG CTG-3'; c-myc lower primer, 5'-TCT TGA CAT TCT CCT CGG TGT CCG AGG ACC T-3'; and c-myc upper primer, 5'-TAC CCT CTC AAC GAC AGC AGC TCG CCC AAC TCC T-3'. RT was carried out in a 10-µl mixture containing total RNA (500 ng of MUC5AC, 50 ng of MUC5B, 250 ng of GAPDH, and 50 ng of c-myc RNAs) and 25 units of Superscript II reverse transcriptase (GIBCO BRL) in buffer containing 10 mM dithiothreitol, 1 mM deoxynucleotide triphosphates (Promega, Madison, WI), 10 units of RNasin (Promega), and 0.2 µM 3' oligonucleotide primer. First-strand synthesis consisted of primer annealing (25°C, 10 min) and template extension (42°C, 45 min) (Biometra Thermocycler, Tampa, FL). Newly synthesized cDNA was amplified by PCR in 50 µl with 0.75 units of Taq polymerase (GIBCO BRL) in Taq buffer (GIBCO BRL) containing 1.5 mM MgCl2 and 0.2 µM 5' primer. The amplification conditions were 20 s at 94°C, 30 s at the annealing temperature (Ta) specific for each primer pair, and 30 s plus 1 s/cycle for n number of cycles (MUC5AC: Ta = 60°C, n = 34; MUC5B: Ta = 59.8°C, n = 30; GAPDH: Ta = 58°C, n = 25; c-myc: Ta = 60°C, n = 30). The specificity of the PCR products was confirmed by dye terminator sequencing with an ABI PRISM cycle sequencing kit (Perkin-Elmer, Foster City, CA).

Quantitation of PCR products. PCR products were quantitated by densitometry as previously described (7). DNA (10 µl) was electrophoresed on a 2% agarose gel containing 0.5 µg/ml of ethidium bromide in 90 mM Tris phosphate-2 mM EDTA buffer. After electrophoresis, DNA was visualized by ultraviolet illumination and photographed (type 665 black and white film, Polaroid). The resulting image was scanned and analyzed by an image-analysis software program (Mocha, Jandel Scientific), and the total intensity (average intensity multiplied by total pixels) of each band was measured. All messages examined amplified exponentially (until saturation) according to the amount of target mRNA in the sample. The relationship between mRNA level and band intensity was determined by a curve-fitting software program (SigmaPlot, Jandel Scientific) and found to be y = a[1 - exp(-bx)] + c, where a is the amplitude of exponential, b is the rate constant, and c is the zero intercept. For each RT-PCR, a serial dilution (1,000-32 ng) of total mRNA was amplified and included on each gel to obtain an internally consistent reference curve. All samples were analyzed in the linear portion of the curve. The relative amount of mRNA was determined by comparing the total intensity of each sample against the standard curve. Samples were analyzed in duplicate, and mucin mRNA levels are expressed as multiple of increase over control level after normalization to GAPDH.

Data analysis. Mucin mRNA levels were determined in duplicate from three to six separate experiments and are presented as means ± SE. Student's t-test was used to determine differences between exposed and control groups. Values with P <=  0.05 were considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of acrolein exposure on mucin mRNA levels. Dose-dependent increases (1.5 ± 0.1-, 2.9 ± 0.6-, and 2.1 ± 0.4-fold) in MUC5AC mRNA levels in NCI-H292 cells were observed after 4-h exposures to 0.01, 0.1, and 1.0 nM acrolein, respectively (Fig. 1). MUC5B mRNA levels were not increased at any concentration tested. After exposure to 100 nM acrolein, mRNA levels encoding MUC5AC, MUC5B, and GAPDH were significantly less than the control levels (Fig. 1). Recovery of total RNA was also reduced at 100 nM (data not shown), indicating that decreased message levels may be a cytotoxic effect of the highest acrolein exposure.


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Fig. 1.   Dose-response relationship of acrolein induction of mucin mRNA levels. Confluent NCI-H292 cells were exposed to acrolein (0.01-100 nM) for 4 h. Total RNA was isolated, and steady-state mucin (MUC5AC) and MUC5B mRNA levels were analyzed by RT-PCR. Top: mucin levels determined by densitometry. Values are means ± SE expressed as multiple of increase over control level (exposed/control); n = 3 experiments. * Significantly different from control value. Bottom: duplicate determinations of mucin RT-PCR products after acrolein exposure. Lane 1, control; lane 2, 0.01 nM; lane 3, 0.1 nM; lane 4, 1.0 nM: lane 5, 10 nM; lane 6, 100 nM. RT-PCR products for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are shown for comparison. Photonegative images are representative of 3 separate experiments.

Agonist effects on mucin mRNA levels. Eicosanoid inflammatory mediators associated with acrolein exposures, PGE2 and 15-HETE significantly increased steady-state MUC5AC mRNA levels in NCI-H292 cells (Fig. 2). Levels of MUC5B mRNA did not change after exposure to these eicosanoids (Fig. 2). Because TNF-alpha may be an important mediator of mucus hypersecretion in human diseases (8, 31) and PMA activates a major signaling pathway in mucus secretion (1), these agonists were also examined for their ability to increase mucin mRNA synthesis. TNF-alpha and PMA also increased MUC5AC but not MUC5B mRNA levels in NCI-H292 cells (Fig. 2). The arachadonic acid metabolites PGE2 or 15-HETE increased MUC5AC expression (2.1 ± 0.2- and 2.0 ± 0.2-fold, respectively) to a lesser extent than either TNF-alpha or PMA (3.0 ± 0.4- and 4.1 ± 1.0-fold, respectively) at the concentrations tested (Fig. 2). These increases are specific because the control mRNA, GAPDH, was not altered by any exposure. Differential regulation of MUC5AC and MUC5B also occurred in another mucin-secreting cell line (Fig. 3). MUC5AC mRNA levels increased in A549 cells exposed to TNF-alpha (2.3 ± 0.2-fold) and PMA (3.1 ± 0.2-fold). Consistent with results from the NCI-H292 cell line, MUC5B mRNA levels were not significantly changed in A549 cells after these exposures (Fig. 3).


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Fig. 2.   Effects of inflammatory mediators and phorbol 12-myristate 13-acetate (PMA) on mucin mRNA levels. Confluent NCI-H292 cells were exposed for 4 h to 10 µM 15-hydroxyeicosatetraenoic acid (15-HETE), 10 µM PGE2, 1.0 nM tumor necrosis factor (TNF)-alpha , and 10 nM PMA. Total RNA was isolated, and MUC5AC and MUC5B mRNA levels were analyzed by RT-PCR. Top: mucin levels determined by densitometry. Values are means ± SE; n = 3 experiments. * Significantly different from control value. Bottom: duplicate determinations of mucin RT-PCR products after agonist exposures. Lane 1, control; lane 2, 15-HETE; lane 3, PGE2; lane 4, TNF-alpha ; lane 5, PMA. RT-PCR products for GAPDH are shown for comparison. Photonegative images are representative of 3 separate experiments.


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Fig. 3.   Effect of TNF-alpha and PMA on MUC5AC and MUC5B mRNA levels in A549 cells. Confluent A549 cells were exposed for 4 h to 1.0 nM TNF-alpha or 10 nM PMA. Total RNA was isolated, and MUC5AC and MUC5B steady-state mRNA levels were analyzed by RT-PCR. Mucin levels were determined by densitometry. Values are means ± SE; n = 3 experiments. * Significantly different from control value.

Under basal conditions, MUC5B could be detected with 10-fold less total RNA and with fewer PCR cycles than those necessary to detect MUC5AC. The abundance of mRNA detected by RT-PCR and the lack of induction by agonists of mucus secretion suggest that MUC5B may be constitutively expressed (Figs. 1-3).

Mechanism of MUC5AC mRNA expression induced by TNF-alpha . To investigate the mechanism of MUC5AC mRNA induction, the time course was initially measured. TNF-alpha stimulated a rapid and transient increase in MUC5AC mRNA levels (Fig. 4). MUC5AC mRNA levels were significantly increased at 1 h, maximal at 2 h, and subsequently decreased, returning to control levels at 16 h posttreatment. MUC5B and GAPDH mRNA levels were not altered by TNF-alpha exposure.


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Fig. 4.   Time course of TNF-alpha -induced MUC5AC mRNA levels. Confluent NCI-H292 cells were exposed for up to 16 h to 1.0 nM TNF-alpha . Total RNA was isolated, steady-state mRNA levels were analyzed by RT-PCR, and mucin mRNA levels were determined by densitometry. RT-PCR products for GAPDH are shown for comparison. Values are means ± SE; n = 6 experiments. * Significantly different from control value.

To determine whether TNF-alpha affected MUC5AC mRNA stability, transcription was pharmacologically inhibited (59) after peak message induction (2 h), and the rate of mRNA decay was determined (Fig. 5). In the absence of TNF-alpha stimulation, the half-life (defined as the time at which 50% of mRNA remained) of MUC5AC in NCI-H292 cells was ~4 h (Fig. 5). After TNF-alpha stimulation, the approximate half-life of MUC5AC mRNA was increased to almost 10 h. This 2.5-fold increase in mRNA half-life could account for most of the 3-fold increase in MUC5AC steady-state mRNA levels observed after TNF-alpha treatment (Fig. 4).


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Fig. 5.   TNF-alpha increases MUC5AC mRNA stability. Confluent NCI-H292 cells were exposed to 1.0 nM TNF-alpha or to PBS alone (control) for 2 h followed by addition of 100 nM 5,6-dichloro-1-beta -D-ribofuranosylbenzimidazole (DRB). Top: MUC5AC mRNA levels determined by densitometry. Values are means ± SE expressed as percent mRNA remaining from time 0; n = 3 experiments. Bottom: duplicate determinations of MUC5AC RT-PCR products after DRB addition to TNF-alpha -treated or untreated cells. Lane 1, 0 h; lane 2, 1 h; lane 3, 2 h; lane 4, 4 h; lane 5, 8 h; lane 6, 16 h. RT-PCR products for c-myc are included as controls. Photonegative images are representative of 3 separate experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Although the mediators and mechanisms controlling mucus secretion have been well studied, less is known regarding the mediators and mechanisms regulating airway mucin synthesis. In this study, we demonstrate that in cells derived from human lung cancers, MUC5AC but not MUC5B is regulated at the mRNA level by acrolein, inflammatory mediators, and PMA. In addition, we found that increased message stability is one of the mechanisms involved in the induction of MUC5AC mRNA after TNF-alpha exposure.

Exposure to several respiratory tract irritants, including acrolein, can cause mucus hypersecretion in animals (23, 29, 33). Recently, Borchers et al. (7) demonstrated that acrolein-induced mucus hypersecretion is associated with increased MUC5AC mRNA and protein synthesis. The present study suggests that acrolein can act directly on epithelial cells to increase mucin mRNA levels (Fig. 1) or indirectly through inflammatory mediators (PGE2 or 15-HETE; Fig. 2) released after exposure (16, 22, 30). High concentrations (10-100 µM) of acrolein increase eicosanoid release from airway epithelial cells (16) or alveolar macrophages (22) and can decrease aldehyde-metabolizing enzymes in the liver (39, 44). A lower concentration (1 µM) can deplete intracellular glutathione (40%) in human bronchial epithelial cells (21, 28). The low acrolein dose (0.01-1.0 nM) inducing MUC5AC mRNA indicates that this end point is more sensitive to the direct effects of acrolein exposure than other end points investigated. At 100 nM acrolein, MUC5AC mRNA levels were decreased. Because glutathione depletion and eicosanoid synthesis occur at doses > 100 nM in other cells and eicosanoid synthesis was not measured in the present study, whether these mechanisms are involved in MUC5AC induction in this experiment are unknown. However, it is possible that small reductions (20%) in glutathione could activate redox-sensitive transcription factors such as nuclear factor-kappa B (43). This possibility is supported by evidence demonstrating nuclear factor-kappa B binding sites within the functional promoter region of MUC5AC (32).

Acrolein exposure in vivo may nonetheless increase MUC5AC mRNA levels through the release of mediators such as eicosanoids. Instillation of PGE1 induces mucous cell development and mucin synthesis in mouse airways (41). Additionally, indomethacin, a prostaglandin synthetase inhibitor, can inhibit mucin synthesis in rat lung after tobacco smoke exposure (46). Although eicosanoid levels were not measured in this study, PGE2 levels increase in the airways of guinea pigs after 1.3 ppm acrolein exposure (30). Acrolein exposure of >= 1.5 ppm increased MUC5AC mRNA and protein in the rat lung (7). In this study, PGE2 and 15-HETE increased MUC5AC mRNA levels in human airway epithelial cells (Fig. 2).

Mucus hypersecretion is a feature of inflammatory airway disorders associated with increased TNF-alpha production, including asthma (8), respiratory syncytial virus infection (6), and cystic fibrosis (42). We therefore examined the regulation of mucin mRNA in response to the proinflammatory cytokine TNF-alpha . TNF-alpha is secreted predominantly by activated macrophages and monocytes and can increase mucus secretion and MUC2 mRNA in human airway epithelial cells (31). We found that TNF-alpha treatment increased MUC5AC but not MUC5B mRNA levels in both NCI-H292 and A549 cells (Figs. 2 and 3). In NCI-H292 cells, TNF-alpha increased MUC5AC mRNA levels over a period of 8 h, reaching maximal levels by 4 h (Fig. 4). This is consistent with reports by other investigators who examined TNF-alpha - and PMA-induced MUC2 mRNA levels in airway (31) and colonic (56) epithelial cells. We did not measure mucin protein levels in this study. However, mucin mRNA induction is relatively short lasting (<= 8 h) compared with the duration of mucin secretion after TNF-alpha exposure (24-72 h) (31). This suggests that subsequent translational and posttranslational mechanisms are involved in the regulation of mucin synthesis and secretion.

One mechanism by which TNF-alpha regulates MUC5AC mRNA levels is by increasing the stability of the transcript (Fig. 5). TNF-alpha can also increase message stability of a glucose transporter gene, GLUT-1 (37, 51). Message stability of GLUT-1 involves induction of proteins that bind to mRNA destabilizing sequences in the 3'-untranslated region (3'-UTR), thus protecting the transcript against nucleolytic cleavage. Approximately 500 bp of the MUC5AC 3'-UTR have been sequenced (9, 38), but whether specific elements within this region control message stabilization remains to be investigated. Although this is the most studied and best understood mechanism, other determinants of mRNA stability have also been demonstrated. The formation of stem-loop structures within the 3'-UTR that serve as protein binding sites and protein binding within the coding region of transcripts also result in an increased message half-life (24). Given that the rate of protein synthesis is directly proportional to the respective cytoplasmic mRNA levels (24), the ability of a cell to increase the stability of such a large message (~14 kb) represents a potentially rapid and efficient means to increase the amount of templates available for translation. This is especially relevant in the context of acute irritation or infection reactions when cells deplete stored mucous granules and must therefore rapidly synthesize nascent mucin proteins.

MUC5AC has been identified in both submucosal glands and surface epithelial cells (4), whereas MUC5B has been localized primarily within submucosal glands (4, 46). Although this study and others (7, 20, 26, 32) indicate that MUC5AC is regulated at the mRNA level, MUC5B mRNA levels were unchanged in both cell lines examined. The lack of MUC5B induction by agonists of mucin synthesis and secretion suggests that its resulting protein may be constitutively expressed. In normal airways, MUC5B could be responsible for maintaining basal levels of mucin synthesis and secretion within secretory cells. In chronic airway diseases, increases in mucin secretion could thereby be achieved through the marked enlargement of the submucosal glands, accompanied by increases in the number of cells involved in MUC5B synthesis. Having an alternative, inducible mucin like MUC5AC enables rapid responses to irritants. In addition, chronic airway diseases often lead to changes in the biochemical composition of airway mucus (14, 25), possibly provided through the differential regulation of inducible and noninducible forms airway mucins.

In summary, we found that MUC5AC but not MUC5B mRNA levels are regulated by acrolein and inflammatory mediators. One mediator, TNF-alpha , regulates MUC5AC by posttranscriptional mechanisms. These results suggest that direct irritant exposure as well as inflammatory mediators contribute to the pathogenesis of obstructive airway disease by increasing mucin mRNA synthesis in airway epithelial cells. The details of the regulation of mucin synthesis now beginning to emerge should increase our understanding of the relative role of mucin genes in airway hypersecretion and provide valuable insights into clinical strategies aimed at alleviating excessive mucus.


    ACKNOWLEDGEMENTS

We thank Drs. Carol Basbaum and Kevin Driscoll for useful advice and suggestions.


    FOOTNOTES

This study was supported by National Institute of Environmental Health Sciences Grants R01-ES-06562, R01-ES-06677, and P30-ES-06096 and National Heart, Lung, and Blood Institute Grant R01-HL-58275.

M. T. Borchers is a recipient of a United States Environmental Protection Agency J. Stara Scholarship Award and University of Cincinnati (OH) Graduate Assistantship. This work is in partial fulfillment of the PhD degree requirements at the University of Cincinnati.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. D. Leikauf, Dept. of Environmental Health, Univ. of Cincinnati, PO Box 670056, Cincinnati, OH 45267-0056 (E-mail: Leikaugd{at}ucmail.uc.edu).

Received 23 September 1998; accepted in final form 23 December 1998.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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