Cystic fibrosis (CF) is an autosomal recessive disorder, the most common lethal genetic disease in Caucasians. Respiratory disease is the major cause of morbidity and mortality. Indeed, 95% of CF patients die of respiratory failure. Pseudomonas aeruginosa, an opportunistic pathogen, chronically infects the lungs of over 85% of CF patients. It is ineradicable by antibiotics and responsible for airway mucus overproduction that contributes to airway obstruction and death. The molecular mechanisms underlying this pathology are unknown. Here we show that P. aeruginosa activates a c-Src-Ras-MEK1͞2-MAPK-pp90rsk signaling pathway that leads to activation of nuclear factor NF-B (p65͞p50). Activated NF-B binds to a B site in the 5-f lanking region of the MUC2 gene and activates MUC2 mucin transcription. These studies bring new insight into bacterialepithelial interactions and more specifically into the molecular pathogenesis of cystic fibrosis. Understanding these signaling and gene regulatory mechanisms opens up new therapeutic targets for cystic fibrosis.Cystic fibrosis (CF) is the most common lethal genetic disease in the Caucasian population (1). The mutation responsible for the disease is in the gene encoding the cystic fibrosis transmembrane regulator (CFTR), a chloride channel. This mutation gives rise to a chain of events involving chronic bacterial lung infection and mucus overproduction. Although there have been significant clues regarding the link between the mutation and chronic infection (2-4), the nature of the link between the mutation and mucus overproduction is completely unknown.We recently showed that bacterial exoproducts up-regulate epithelial MUC2 mucin transcription (5). This suggests that CF-associated mucin overproduction occurs secondary to lung infection, a sine qua non of advanced disease. The molecular mechanisms underlying mucin induction are unknown. In the present study, we performed experiments to examine these mechanisms. Results showed that Pseudomonas aeruginosa, a common CF pathogen, activates the MUC2 mucin gene transcription by activation of a Src-dependent Ras-MEK1͞2-ERK1͞2-pp90rsk-NF-B pathway. These studies provide insight into bacterial-host epithelial interactions and open up new therapeutic targets for CF. MATERIALS AND METHODSReagents. P. aeruginosa lipopolysaccharide (LPS) from serotype 10 was purchased from Sigma. PP1, PD98059, and caffeic acid phenethyl ester (CAPE) were purchased from Calbiochem.Bacterial Strains and Culture Conditions. The P. aeruginosa strains used in these studies were grown in M9 medium with aeration at 37°C to late logarithmic phase. The broth cultures were then centrifuged at 10,000 rpm in a Sorvall RC5C for 50 min. The supernatants containing bacterial exoproducts were sterilized by passage through a 0.22-m polymer filter (Corning) and were then kept at Ϫ80°C until use. Bacterial culture supernatants were added to epithelial cell culture medium at a 1:4 dilution ratio.Cell Culture. Two MUC2-expressing epithelial cell lines were used in these...

An unresolved question in cystic fibrosis (CF) research is how mutations of the CF transmembrane conductance regulator, a Cl ion channel, cause airway mucus obstruction leading to fatal lung disease. Recent evidence has linked the CF transmembrane conductance regulator mutation to the onset and persistence of Pseudomonas aeruginosa infection in the airways, and here we provide evidence directly linking P. aeruginosa infection to mucus overproduction. We show that P. aeruginosa lipopolysaccharide profoundly upregulates transcription of the mucin gene MUC 2 in epithelial cells via inducible enhancer elements and that this effect is blocked by the tyrosine kinase inhibitors genistein and tyrphostin AG 126. These findings improve our understanding of CF pathogenesis and suggest that the attenuation of mucin production by lipopolysaccharide antagonists and tyrosine kinase inhibitors could reduce morbidity and mortality in this disease.

Cells dividing at the time of carcinogen exposure are at particular risk for neoplasia. Tobacco smoke contains numerous carcinogens, and we find that smoke, in the absence of exogenous growth factors, is capable of stimulating cell proliferation. The smoke-triggered mechanism includes the generation of oxygen radicals, which in turn stimulate tumor necrosis factor ␣-converting enzyme (a disintegrin and metalloproteinase (ADAM) 17) to cleave transmembrane amphiregulin, a ligand for the epidermal growth factor receptor (EGFR). The binding of amphiregulin to EGFR then stimulates proliferation of lung epithelial cells. These results shed light on the pathogenesis of lung cancer, suggest novel drug targets for the reduction of cancer risk in smokers, and provide insight into how EGFR integrates responses to diverse noxious stimuli.

Mucus hypersecretion and plugging of lower respiratory tract airways contributes to the morbidity and mortality associated with asthma. Interleukin (IL)-4 plays a putative role in some forms of asthma. Thus, transgenic mice that overexpress murine IL-4 selectively within the lung were used to study the effect of IL-4 on mucus glycoprotein gene expression and mucin release. Histologic examination of lung sections from IL-4 mice revealed that nonciliated epithelial cells from conducting airways were hypertrophic, due at least in part to the accumulation of mucus glycoprotein. The cytoplasm of these cells stained positively for glycoproteins using mucicarmine, alcian blue (AB), and periodic acid-Schiff (PAS). Ciliated cells were also enlarged but did not show any mucin-specific staining. Inclusion granules typically found in nonciliated (Clara) cells of control mice were absent in the IL-4 transgenic mice. Northern blot analysis of total RNA from lung tissue revealed that the expression of the MUC5AC, but not MUC2, mucin gene was distinctly upgraded in IL-4 transgenic mice compared to transgene-negative controls. In addition, a 5- to 10-fold increase in AB- and PAS-positive material was found in lavage fluid from IL-4 overexpressing mice compared to transgene-negative controls. Thus, the overexpression of IL-4 locally within the lung enhances mucus glycoprotein synthesis by altering gene expression, results in the accumulation of mucus glycoprotein in nonciliated epithelial cells, and induces the release of mucus into the airway lumen. We therefore hypothesize that the overproduction of mucus seen in some patients with asthma may be a direct result of the action of IL-4 within the inflamed lung.

Bacterial infection of the lung is associated with mucin overproduction. In partial explanation of this phenomenon, we recently reported that supernatant from the Gram-negative organism Pseudomonas (P.) aeruginosa contained an activity that upregulated transcription of the MUC 2 mucin gene [J.-D. Li, A. Dohrman, M. Gallup, S. Miyata, J. Gum, Y. Kim, J. Nadel, A. Prince, C. Basbaum, Transcriptional activation of mucin by P. aeruginosa lipopolysaccharide in the pathogenesis of cystic fibrosis lung disease, Proc. Natl. Acad. Sci. U.S.A., 94 (1997) 967-972]. The purpose of the present study was to determine whether mucin genes other than MUC 2 are so regulated and whether Gram-positive organisms also contain mucin stimulatory activity. Results from in situ hybridization and RNase protection assays showed that P. aeruginosa upregulates MUC 5AC as well as MUC 2 in both bronchial explants and cultured airway epithelial cells. The upregulation of both genes by P. aeruginosa can be mimicked by lipopolysaccharide (LPS) and can be blocked by the tyrosine kinase inhibitor genistein. In addition, both genes are upregulated by a variety of Gram-positive as well as Gram-negative organisms showing the same rank order of potency. These data indicate the existence of a general mechanism by which epithelial cells respond to the presence of bacteria by increasing mucin synthesis.

A hallmark of asthma is mucin overproduction, a condition that contributes to airway obstruction. The events responsible for mucin overproduction are not known but are thought to be associated with mediators of chronic inflammation. Others have shown that T-helper 2 (Th2) lymphocytes are required for mucous cell metaplasia, which then leads to mucin overproduction in animal models of allergy. We hypothesized that Th2 cell mediators are present in asthmatic airway fluid and directly stimulate mucin synthesis in airway epithelial cells. Results in cultured airway epithelial cells showed that samples of asthmatic fluid stimulated mucin (MUC5AC) synthesis severalfold more potently than non-asthmatic fluid. Consistent with this, lavage fluid from the airways of allergen-challenged dogs stimulated mucin synthesis severalfold more potently than that from non-allergen-challenged dogs. Fractionation of dog samples revealed 2 active fractions at <10 kDa and 30-100 kDa. Th2 cytokines in these molecular weight ranges are IL-9 (36 kDa), IL-5 (56 kDa), and IL-13 (10 kDa). Antibody blockade of ligand-receptor interaction for IL-9 (but not IL-5 or IL-13) inhibited mucin stimulation by dog airway fluid. Furthermore, recombinant IL-9, but not IL-5 or IL-13, stimulated mucin synthesis. These results indicate that IL-9 may account for as much as 50-60% of the mucin-stimulating activity of lung fluids in allergic airway disease.

To obtain gene regulatory sequence for the mucin gene MUC5AC, we have isolated the MUC5AC amino terminus cDNA and 5-flanking region. This was possible through the use of rapid amplification of cDNA endspolymerase chain reaction (RACE-PCR) in which the 5 sequence of the human gastric mucin cDNA HGM-1 (1) was used to design the first MUC5AC-specific primer. Primers for subsequent rounds of RACE were designed from the 5-ends of amplified RACE products. After five rounds of RACE-PCR, we could no longer generate upstream extensions of the cDNA and hypothesized that we had reached the 5-end. Primer extension and RNase protection analysis confirmed this. Combined nucleotide sequence for the RACE-PCR products was 3.3 kb with an open reading frame encoding 1100 amino acids. A putative translation start site was found at nucleotide ؉48. This was followed by a 45 nucleotide putative signal sequence. This amino-terminal sequence contains no tandem repeats but is >60% similar to the amino-terminal nucleotide sequence of MUC2. The positions of cysteine residues in this MUC2-similar region are almost 100% conserved between the two genes. Northern analysis showed expression of cognate RNA in the stomach and airway but not muscle and esophagus. This pattern was the same as that obtained using previously reported 3-MUC5AC sequences. We have cloned approximately 4 kb of genomic DNA upstream of the transcription start site and have sequenced 1366 nucleotides containing a TATA box, a CACCC box, and putative binding sites for NFB and Sp 1. Within 4 kb of the transcription start site are elements mediating transcriptional up-regulation in response to bacterial exoproducts.Mucin is a glycoprotein secreted from epithelial cells at many body surfaces. In the airways, mucin interacts with cilia to trap and clear pathogens and irritants. This mucociliary mechanism is impaired when mucin is produced excessively as in cystic fibrosis, chronic bronchitis, and asthma. Mucociliary impairment leads to airway mucus plugging, which promotes chronic infection, airflow obstruction, and sometimes death.Nine mucin genes are known to be expressed in man: MUC5AC, MUC5B,. The mRNAs encoding two of them, MUC2 and MUC5AC, have been shown to be up-regulated in cystic fibrosis airways (13,14) 1 and likely contribute to the airway mucus plugging characteristic of this disease. Insofar as DNA-RNA transcription is controlled by mechanisms amenable to pharmaceutical intervention, an understanding of mucin transcription may suggest ways of inhibiting mucin overproduction.Both MUC2 and MUC5AC map to chromosome 11p15.5 and may have arisen from a common ancestral gene. The structure of MUC2 is known. Its central region, comprising Ͼ50% of the polypeptide, contains two tandem repeat sequences rich in threonine, serine, and proline (4, 17); this is flanked up-and downstream by cysteine-rich regions (17, 18). The threonine and serine residues represent O-glycosylation sites, whereas the cysteine residues are thought to mediate intermolecular interactions underlying mu...

In smokers' lungs, excessive mucus clogs small airways, impairing respiration and promoting recurrent infection. A breakthrough in understanding this pathology was the realization that smoke could directly stimulate mucin synthesis in lung epithelial cells and that this phenomenon was dependent on the cell surface receptor for epidermal growth factor, EGFR. Distal steps in the smoke-triggered pathway have not yet been determined. We report here that the predominant airway mucin (MUC5AC) undergoes transcriptional up-regulation in response to tobacco smoke; this is mediated by an AP-1-containing response element, which binds JunD and Fra-2. These transcription factors require phosphorylation by upstream kinases JNK and ERK, respectively. Whereas ERK activation results from the upstream activation of EGFR, JNK activation is chiefly EGFR-independent. Our experiments demonstrated that smoke activates JNK via a Src-dependent, EGFRindependent signaling cascade initiated by smoke-induced reactive oxygen species. Taken together with our earlier results, these data indicate that the induction of mucin by smoke is the combined effect of mutually independent, reactive oxygen species activation of both EGFR and JNK.The primary cause of morbidity in chronic bronchitis is mucin overproduction, a phenomenon for which the molecular pathogenesis is unknown. Inflammatory cells are abundant in smokers' airways (1-3) and are capable of stimulating mucin production (4 -7), suggesting that at least some of the excessive mucin in smokers' lungs is secondary to inflammation.In addition, however, smoke itself can induce mucin synthesis in lung cells (8,9). The question of how this occurs is complex in that smoke, a composite of irritant molecules including acetaldehyde, hydroquinone, formaldehyde, benzo-[a]pyrene, cresol, nicotine, catechol, acrolein, coumarin, anthracene, nitrogen oxides, and heavy metals (10, 11) may act on lung epithelial cells in diverse ways. For example, the induction of cytochrome P450 by tobacco smoke (12) is mediated by binding of the aryl hydrocarbon nuclear receptor to a dioxin response element in the 5Ј-flank of the gene, but the induction of the ␥-glutamylcysteine synthetase heavy subunit (␥-GCS-HS) gene is mediated by the binding of a c-Jun/c-Jun homodimer to an AP-1-like response element (13).Previous reports have implicated the receptor for epidermal growth factor (EGFR) 1 in the induction of mucin gene MUC5AC by smoke (9). Consistent with a role for EGFR in mucin induction, an EGF response element has been identified 200 bp upstream of the MUC5AC gene (14). The response of this element to EGFR ligands EGF and transforming growth factor-␣ is mediated by Sp1. One might predict from these data that the induction of MUC5AC by smoke would depend on interaction between the EGF response element at Ϫ200 bp and Sp1.In contrast, in the present study we show that MUC5AC is controlled principally by a smoke response element ϳ3 kb upstream of the EGF response element. This element is AP-1-dependent and is bound by Ju...