Mucin overproduction is a hallmark of nontypeable Haemophilus influenzae (NTHi) infections. The molecular mechanisms underlying up-regulation of mucin in NTHi infections especially during the initial phase remain unknown. Here we show that P6, a 16-kDa outer membrane lipoprotein well conserved in NTHi, up-regulates MUC5AC mucin gene transcription in vitro and in vivo. Moreover, P6 induces MUC5AC transcription via TLR2-MyD88-IRAK1-TRAF6-TAK1-dependent p38 MAPK-AP1 and IKKbeta-IkappaBalpha-NF-kappaB signaling pathways. This study may bring new insights into the molecular pathogenesis of NTHi-induced infections and lead to novel therapeutic intervention for inhibiting mucin overproduction in patients with NTHi infections.

MUC2 is a secretory mucin normally expressed by goblet cells of the intestinal epithelium. It is overexpressed in mucinous type colorectal cancers but down-regulated in colorectal adenocarcinoma. Phorbol 12-myristate 13-acetate (PMA) treatment of colon cancer cell lines increases MUC2 expression, so we have undertaken a detailed analysis of the effects of PMA on the promoter activity of the 5'-flanking region of the MUC2 gene using stably and transiently transfected promoter reporter vectors. Protein kinase C inhibitors (bisindolylmaleimide, calphostin C) and inhibitors of mitogen-activated protein/extracellular signal regulated kinase kinase (MEK) (PD98059 and U0126) suppressed up-regulation of MUC2. Sre tyrosine kinase inhibitor PP2, a protein kinase A inhibitor (KT5720), and a p38 inhibitor (SB 203580) did not affect transcription. Western blotting and reverse transcription-PCR analysis confirmed these results. In addition, co-transfections with mutants of Ras, Raf, and MEK showed that the induction of MUC2 promoter activity by PMA required these three signaling proteins. Our results demonstrate that PMA activates protein kinase C, stimulating MAP kinase through a Ras- and Raf-dependent mechanism. An important role for nuclear factor kappaB (NF-kappaB) was also demonstrated using the inhibitor caffeic acid phenethyl ester and electrophoretic mobility shift assays. Such identification of pathways involved in MUC2 upregulation by PMA in the HM3 colon cancer cell line may serve as a model for the effects of cytokines and growth factors, which regulate MUC2 expression during the progression of colorectal cancer.

Nontypeable Haemophilus influenzae(NTHi) is an important human pathogen that causes chronic otitis media with effusion (COME) in children and exacerbation of chronic obstructive pulmonary disease (COPD) in adults. Mucin overproduction, a hallmark of both diseases, has been shown to directly cause conductive hearing loss in COME and airway obstruction in COPD. The molecular mechanisms underlying mucin overproduction in NTHi infections still remain unclear. Here, we show that NTHi strongly up-regulatesMUC5AC mucin transcription only after bacterial cell disruption. Maximal up-regulation is induced by heat-stable bacterial cytoplasmic proteins, whereas NTHi surface membrane proteins induce only moderate MUC5AC transcription. These results demonstrate an important role for cytoplasmic molecules from lysed bacteria in the pathogenesis of NTHi infections, and may well explain why many patients still have persistent symptoms such as middle ear effusion in COME after intensive antibiotic treatment. Furthermore, our results indicate that activation of p38 mitogen-activated protein kinase is required for NTHi-induced MUC5AC transcription, whereas activation of phosphoinositide 3-kinase-Akt pathway leads to down-regulation of NTHi-induced MUC5AC transcription via a negative cross-talk with p38 mitogen-activated protein kinase pathway. These studies may bring new insights into molecular pathogenesis of NTHi infections and lead to novel therapeutic intervention for COME and COPD.

Human and animal exposure to particulate air pollution is correlated with airway mucus hypersecretion and increased susceptibility to infection. Seeking clues to the mechanisms underlying this pathology, we examined the effect of the particulate air pollutant residual oil fly ash (ROFA) on production of the major component of mucus, mucin, and the major antibacterial protein of the respiratory tract, lysozyme. We found that following in vitro exposure to ROFA, epithelial cells showed an increase in mucin (MUC5AC) and lysozyme (LYS) steady state mRNA. This upregulation was controlled at least partly at the level of transcription as shown by reporter assays. Experiments testing the ability of the major components of ROFA to mimic these effects showed that vanadium, a metal making up 18.8% by weight, accounted for the bulk of the response. A screen of signaling inhibitors showed that MUC5AC and LYS induction by ROFA are mediated by dissimilar signaling pathways, both of which are, however, phosphotyrosine dependent. Recognizing that the ROFA constituent vanadium is a potent tyrosine phosphatase inhibitor and that mucin induction by pathogens is phophotyrosine dependent, we suggest that vanadium-containing air pollutants trigger disease-like conditions by unmasking phosphorylation-dependent pathogen resistance pathways.

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 ends-polymerase 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 ofMUC2. The positions of cysteine residues in thisMUC2-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′-MUC5ACsequences. 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 NFκB and Sp 1. Within 4 kb of the transcription start site are elements mediating transcriptional up-regulation in response to bacterial exoproducts.

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 respond to changes in their microenvironment by altering their cell surface and extracellular matrix proteins. Rapid and irreversible changes in these proteins are possible through their degradation or activation by proteolysis. By focalizing the proteolytic events at or near the cell surface, these processes can be effective even in the presence of high concentrations of inhibitors. Evidence is emerging that secreted and transmembrane matrix metalloproteinases, metalloproteinases of the adamalysin and astacin (tolloid) families, and serine proteinases are crucial in development, differentiation, cell motility and invasion, and cell—extracellular decisions.

BACKGROUND/AIMS:Several studies have reported Northern blot data showing that mucin is expressed in a tissue-specific manner. To determine whether expression is limited to specific cell types within these tissues requires histological analysis.METHODS:Both immunocytochemistry and in situ hybridization were used to identify cell types expressing the MUC2 and MUC3 mucins in the human small intestine, colon, and colon carcinoma.RESULTS:In the normal small intestine and colon, an antibody recognizing the MUC2 apomucin stained goblet cells. In contrast, an antibody recognizing the MUC3 apomucin stained both goblet and absorptive cells. Consistent with this, in situ hybridization showed MUC2 messenger RNA (mRNA) only in goblet cells and MUC3 mRNA in both goblet and absorptive cells. In several samples of moderately well-differentiated colon cancer, MUC2 and MUC3 showed distinct patterns of expression, but the expression level of each was reduced compared with levels in normal tissue; there was considerable tumor-to-tumor and cell-to-cell variability using both mucin antibodies and complementary DNA probes.CONCLUSIONS:Individual mucin genes have distinct patterns of expression within mucin-producing tissues, suggesting that the various mucin gene products play distinct functional roles.

To obtain cDNAs for analysis of mucin gene transcription in rat models of human disease, we screened a rat intestinal cDNA library in lambda ZAPII using an upstream non-tandem repeat cDNA fragment of the human MUC 2 gene (Gum, J., Hicks, J., Toribara, N., Rothe, E., Lagace, R., and Y., K. (1992) J. Biol. Chem. 267, 21375-21383). Three cDNAs, 1-1, 8-1, and 21-1, were isolated. A translation start site was found in cDNA 21-1. Combined nucleotide sequence for the three cDNAs contained an open reading frame spanning 4546 base pairs. This amino-terminal sequence contains a non-tandem repeat domain enriched in cysteine (1391 residues) followed by an irregular tandem repeat domain (122 residues). Identity with the human gene is about 80% in the non-tandem repeat domain and about 38% in the irregular tandem repeat domain. Primer extension and S1 nuclease protection analysis indicate a transcription start site at 28 base pairs upstream of translation initiation. Northern analysis showed expression of cognate RNA in the intestine and airway but not heart and spleen. The cDNAs have been used to isolate the gene promoter, the structure of which should yield clues to the regulation of mucin expression in rat models of human disease.

To determine whether markers of mucus secretion can be quantified in airway lining fluid from asthmatic and from healthy subjects, we measured levels of a mucin-like glycoprotein (MLG) and lactoferrin in sputum induced by inhalation of hypertonic (3%) saline in 18 asthmatic and in 10 healthy subjects. Because DNA, like mucin, contributes to the viscosity of airway secretions, we also measured DNA levels in the induced sputum samples. To control for the presence of saliva in sputum, we also analyzed saliva samples from all subjects. The entire sputum sample and the saliva sample were reduced using dithiotreitol, and biochemical analysis was performed on supernatants obtained after centrifugation. We found that induced sputum from asthmatic subjects had higher levels of MLG [2,574.4 +/- 907.8 (mean +/- SEM) versus 562.2 +/- 90.5 micrograms/ml, p < 0.007] and DNA (7.1 +/- 1.6 versus 3.6 +/- 0.6 micrograms/ml, p < 0.05), but the difference in lactoferrin levels failed to reach statistical significance. However, in the subgroup of asthmatic subjects who gave a history of sputum production (n = 9), lactoferrin levels were higher than in the healthy control subjects (118.9 +/- 46.3 versus 35.2 +/- 6.5 micrograms/ml, p < 0.05). The very low levels of MLG, DNA, and lactoferrin measured in saliva were not significantly different in asthmatic subjects from those in healthy subjects. We conclude that measurement of markers of mucus secretion in induced sputum is feasible in asthmatic and healthy subjects, and it reveals abnormally high markers of mucus secretion in subjects with stable asthma.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of adenosine 3',5'-cyclic monophosphate (cAMP) and protein phosphorylation during beta-adrenergic receptor stimulation of bovine tracheal gland serous cells was investigated in vitro. Isoproterenol, a beta-adrenergic agonist, increased the secretion of 35S-labeled molecules. Intracellular cAMP levels were increased within 1 min after stimulation of bovine tracheal gland serous cells with isoproterenol. The dose-response relationship for isoproterenol-stimulated generation of cAMP correlated with the dose-response relationship for isoproterenol-stimulated secretion of 35S-labeled molecules. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine potentiated both isoproterenol-evoked secretion of 35S-labeled molecules and the production of intracellular cAMP, and the beta-adrenergic receptor antagonist propranolol completely blocked both effects. The secretory response of the cells to isoproterenol could be mimicked by the cAMP analogues 8-bromoadenosine 3',5'-cyclic monophosphate and dibutyryl adenosine 3',5'-cyclic monophosphate. Activity of cAMP-dependent kinase was measured in soluble and particulate cell extracts. cAMP effected the state of phosphorylation of proteins associated with the soluble but not the particulate fraction. These studies are consistent with the hypothesis that beta-adrenergic stimulation of secretion from bovine tracheal gland serous cells occurs via a cAMP-mediated pathway and that one of the molecular events in this pathway is cAMP-dependent protein phosphorylation.

The extracellular matrix has been shown to influence the differentiation of epithelial cells. To identify cues from the extracellular matrix controlling the differentiation of tracheal gland serous cells, we examined the effects of culturing these cells on various extracellular matrix proteins. Bovine tracheal gland (BTG) serous cells attached to Type IV collagen (COL IV), laminin (LM), and fibronectin (FN) in a concentration-dependent manner. Morphologic analysis showed that cells formed confluent monolayers on COL IV or LM, whereas on FN, cells formed birefringent spheres. Metabolic labeling experiments showed that [35S]methionine-labeled protein bands at 68, 105, and 120 kD were prominent when cells were grown on COL IV or LM, but were lost or reduced when the cells were grown on FN. COL IV also enhanced the expression of proteins at 14, 16.5, 18, and 21.5 kD. Attachment to all substrates was inhibited by an antibody directed against beta 1 integrins. This antibody precipitated several integrin heterodimers from a BTG cell membrane extract, caused partial retraction of cells from all substrates, and strongly suppressed the expression of COL IV- and LM-dependent proteins. Control experiments indicated that the latter did not require conspicuous changes in cell shape. These results show that some biochemical properties of serous cells are regulated by integrin-mediated effects of extracellular matrix proteins in vitro and suggest that similar regulation may occur during normal development and remodeling of the glands in vivo.