Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells.

Chronic neutrophil-predominant inflammation and hypersecretion of mucus are common pathophysiological features of cystic fibrosis, chronic bronchitis, and viral- or pollution-triggered asthma. Neutrophils release elastase, a serine protease, that causes increased mucin production and secretion. The molecular mechanisms of elastase-induced mucin production are unknown. We hypothesized that as part of this mechanism, elastase upregulates expression of a major respiratory mucin gene, MUC5AC. A549, a human lung carcinoma cell line that expresses MUC5AC mRNA and protein, and normal human bronchial epithelial cells in an air-liquid interface culture were stimulated with neutrophil elastase. Neutrophil elastase increased MUC5AC mRNA levels in a time-dependent manner in both cell culture systems. Neutrophil elastase treatment also increased MUC5AC protein levels in A549 cells. The mechanism of MUC5AC gene regulation by elastase was determined in A549 cells. The induction of MUC5AC gene expression required serine protease activity; other classes of proteases had no effect on MUC5AC gene expression. Neutrophil elastase increased MUC5AC mRNA levels by enhancing mRNA stability. This is the first report of mucin gene regulation by this mechanism.

Tumor necrosis factor-alpha stimulates mucin secretion and cyclic GMP production by guinea pig tracheal epithelial cells in vitro.

Tumor necrosis factor (TNF)-alpha, a pluripotent cytokine implicated in the pathogenesis of airway inflammation, has been shown to provoke hypersecretion of mucin by airway epithelial cells in vitro. In this study, we investigated potential signaling pathways mediating TNF-alpha-induced mucin secretion using guinea pig tracheal epithelial (GPTE) cells in air-liquid interface culture. Exogenously applied TNF-alpha (human recombinant) stimulated mucin secretion in a concentration-dependent manner, with maximal effects at 10 to 15 ng/ml (286 to 429 U/ml). The pathway of stimulated secretion appeared to involve generation of intracellular nitric oxide (NO), activation of soluble guanylate cyclase (GC-S), production of cyclic guanosine monophosphate (cGMP), and activation of cGMP-dependent protein kinase (PKG). TNF-alpha increased production of nitrite and nitrate by GPTE cells; both mucin secretion and cGMP production were attenuated by NG-monomethyl-L-arginine (1 mM), a competitive inhibitor of nitric oxide synthase (NOS), or by the GC-S inhibitor LY83583 (50 microM); and mucin secretion in response to TNF-alpha or to the cGMP analogue dibutyryl cGMP (100 and 500 microM) was attenuated by the specific PKG inhibitor KT5823 (1 microM). Increased mucin secretion and increased cGMP production in response to TNF-alpha both appeared to be mediated by a phospholipase C that hydrolyzes phosphatidylcholine (PC-PLC), and by protein kinase C (PKC), since both responses were attenuated by either D609 (10 and 20 microg/ml), a specific PC-PLC inhibitor, or by each of three PKC inhibitors: Calphostin C (0.3 and 0.5 microM), bisindoylmaleimide (GF 109203X, Go 6850; 20 nM), or Ro31-8220 (10 microM). Collectively, the results suggest that TNF-alpha stimulates secretion of mucin by GPTE cells via a mechanism(s) dependent on PC-PLC and PKC, and involving activation of NOS, generation of NO, production of cGMP, and activation of PKG.

Respiratory carcinoma cell lines. MUC genes and glycoconjugates.

Lung carcinoma cell lines are being used in many laboratories to study various airway epithelial functions, including mucin gene expression. To identify model systems for investigating regulation of MUC5/5AC gene expression and secretion of MUC5/5AC mucins in airway epithelial cells, we evaluated the expression of several mucin genes in six carcinoma cell lines of respiratory tract origin. RNA was extracted from A549, Calu-3, NCI H292, Calu-6, RPMI 2650, and A-427 cells; MUC1, MUC2, MUC4, MUC5/5AC, and MUC5B messenger RNA (mRNA) expression was determined. By Northern analyses, all cell lines expressed MUC1 mRNA, whereas MUC2 mRNA was not detectable in any of the cell lines. RPMI 2650 cell lines expressed only MUC1 mRNA. NCI-H292 cells expressed MUC4 and low levels of MUC5/5AC mRNA. Calu-3 and A549 cells expressed MUC5/5AC mRNA; A549 cells also expressed MUC5B mRNA. Glycoconjugates secreted by lung carcinoma cells were also examined. By wheat germ lectin analysis, Calu-3, H292, and A549 cells secreted high molecular weight glycoproteins having N-acetylglucosamine and/or sialic acid moieties. Western blot analyses with an anti-MUC5:TR-3A antibody demonstrated that Calu-3 and A549 cells secreted MUC5/5AC mucins. All six carcinoma cell lines secreted large, radiolabeled, sulfated macromolecules; the majority were proteoglycans that were digested by hyaluronidase. However, Calu-3 cells also secreted sulfated high molecular-weight glycoproteins that were immunoprecipitated by anti-MUC5:TR-3A antibody. These studies demonstrated that Calu-3 and A549 cell lines expressed high and moderate amounts of MUC5/5AC mRNA and MUC5/5AC mucins, whereas H292 cells expressed lesser amounts. These cell lines should prove useful for studies of MUC5/5AC gene expression and MUC5/5AC biosynthesis, trafficking, and secretions in airway epithelial cells.

Mucin gene expression in ovarian cancers.

Ovarian cancer is a highly lethal disease with metastases present in the majority of patients at the time of diagnosis. The molecular mechanisms underlying the metastatic process of this cancer are not well understood. One family of cell-associated and secreted glycoproteins, the mucin glycoproteins, has been implicated in events leading to metastasis of several epithelial cancers including gastrointestinal and lung cancers. The purpose of this study was to characterize mucin gene expression in ovarian cancers and relate expression to tumor histology, stage, and patient survival. RNA was isolated from 29 epithelial ovarian cancers, 1 neuroendocrine carcinoma, 3 mixed mesodermal tumors, and two transformed, yet nonmalignant, ovarian epithelial cell lines. The expression of mucin genes, MUC1, 2, 3, 4, 5AC and 5B, was determined by northern analyses. Epithelial ovarian cancers expressed several mucins including MUC1, 2, 4, and 5AC; MUC3 and 5B were rarely expressed. In contrast, the transformed nonmalignant ovarian epithelial cell lines expressed only MUC1 and 5AC. Although there was no correlation of mucin expression with tumor histology, there was a significant decrease in expression of MUC3 and MUC4 with increasing cancer stage (P < 0.05). In addition, a trend toward improved patient survival occurred with increased expression of MUC4. These observations suggest a relationship between mucin gene expression and the metastatic process in epithelial ovarian cancers. Additional investigation of MUC3 and MUC4 in ovarian cancers may lead to new approaches for early detection and therapy.

The role of reactive oxygen and nitrogen species in airway epithelial gene expression.

The body first encounters deleterious inhaled substances, such as allergens, industrial particles, pollutants, and infectious agents, at the airway epithelium. When this occurs, the epithelium and its resident inflammatory cells respond defensively by increasing production of cytokines, mucus, and reactive oxygen and nitrogen species (ROS/RNS). As inflammation in the airway increases, additional infiltrating cells increase the level of these products. Recent interest has focused on ROS/RNS as potential modulators of the expression of inflammation-associated genes important to the pathogenesis of various respiratory diseases. ROS/RNS appear to play a variety of roles that lead to changes in expression of genes such as interleukin-6 and intercellular adhesion molecule 1. By controlling this regulation, the reactive species can serve as exogenous stimuli, as intercellular signaling molecules, and as modulators of the redox state in epithelial cells. Unraveling the molecular mechanisms affected by ROS/RNS acting in these capacities should aid in the understanding of how stimulated defense mechanisms within the airway can lead to disease.

Mucin gene expression (MUC1, MUC2, and MUC5/5AC) in nasal epithelial cells of cystic fibrosis, allergic rhinitis, and normal individuals.

The early pathogenic events in cystic fibrosis (CF) include colonization of Pseudomonas in the lung, airway inflammation, and mucus hypersecretion with airway obstruction. The primary mechanisms leading to chronic infection and inflammation are not well understood. One possible explanation for this cascade of events is increased or altered expression of one or more mucin (MUC) genes by CF cells in the respiratory tract. We compared expression levels of three mucin genes, MUC1, MUC2, and MUC5/5AC, known to be expressed in the respiratory tract of CF, allergic rhinitis, and normal individuals. Mucin transcript levels in nasal epithelial cells free from inflammation were quantitated by an MUC mRNA slot-blot method. This study revealed three major findings: (1) MUC5/5AC mRNA was expressed at five- to tenfold greater levels than MUC2 or MUC1 for all subjects. (2) MUC2 mRNA levels were similar among all subject groups. (3) In CF subjects, there was a trend toward reduced MUC5/5AC expression. When normalized to the levels of MUC2 expression in individual specimens, MUC5/5AC expression was reduced significantly in CF cells compared with normal cells. These data suggest that mucin gene expression is altered in noninflamed CF nasal cells.

Quantitation of mucin mRNA in respiratory and intestinal epithelial cells.

Mucin glycoproteins (mucins) are the major macromolecular constituents of mucus gels in mammalian respiratory, gastrointestinal, and reproductive tracts. Disorders of mucin glycosylation, which may result from either abnormal post-translational processing or differences in mucin protein gene expression, have been indicated in several diseases. Quantitation of mucin gene expression has been hindered by two features of human mucin genes: variable numbers of tandemly repeating nucleotides per mRNA molecule and polydisperse mRNA transcripts. We report here a method to quantitate mucin mRNA levels in epithelial cells and have evaluated three mucin genes, MUC1, MUC2, and MUC5, which are expressed in respiratory epithelium. The method uses the 3' non-tandem repeat mucin cDNA sequences, as they were shown to have a single-size transcript when amplified by the polymerase chain reaction, consistent with a one-to-one relationship with the mRNA molecule. The 3' non-tandem repeat cDNA sequences were cloned and transcribed in vitro to prepare complementary RNA (cRNA) standards. By comparison to a cRNA standard curve, mucin gene expression was evaluated in colon adenocarcinoma, pancreatic adenocarcinoma, and transformed respiratory epithelial cells and in nasal polyp tissue by slot blot analysis. CFPAC-1, a pancreatic adenocarcinoma cell line, expressed the highest MUC1 transcript levels. Colon adenocarcinoma cell lines varied in MUC2 expression levels, and one colon adenocarcinoma cell line, HT-29, had higher levels of MUC5 than MUC2. Nasal polyp tissue expressed more MUC5 mRNA than MUC1 or MUC2 mRNA. This mucin mRNA slot blot method provides a quantitative method for investigating the regulation of mucin gene expression in health and disease.

Purification and characterization of GDP-L-fucose-N-acetyl beta-D-glucosaminide alpha 1----6fucosyltransferase from cultured human skin fibroblasts. Requirement of a specific biantennary oligosaccharide as substrate.

GDP-L-fucose-N-acetyl-beta-D-glucosaminide alpha 1----6fucosyltransferase which catalyzes the transfer of fucose from GDP-L-fucose to the asparagine-linked N-acetyl-beta-D-glucosamine of N-linked glycoproteins has been purified 37,000-fold from cultured human skin fibroblasts. The Km values for the substrate asialoagalactotransferrin glycopeptide, and GDP-L-fucose were 66 and 4.2 microM, respectively. The Vmax was 1.4 mumols/mg/min. The key step in enzyme purification was affinity chromatography using the immobilized substrate asialoagalactotransferrin glycopeptide-CH-Sepharose. The affinity-purified enzyme had a minimum substrate requirement for a biantennary oligosaccharide with GlcNAc in terminal position, having a Km value of 55 microM. It was heretofore unexpected that the oligosaccharide would serve as substrate, since the site of enzyme activity is GlcNAc-1-linked to Asn. Although the presence of amino acids on this oligosaccharide enhanced the activity 3-fold, it is proposed that this may be the result of an alpha/beta anomeric mixture (2:1) of oligosaccharide used in these studies with only the beta anomer active as substrate. The implication is that the amino acid is required only to retain the beta anomeric position of the substrate. Removal of GlcNAc or addition of Gal to either the oligosaccharide or glycopeptide destroyed the ability to serve as substrates. In addition, di-N-acetylchitobiose, tri-N-acetylchitotriose and GlcNAc beta 1----Asn were nonpermissible substrates. This rigid substrate requirement is unique among fucosyltransferases thus far reported, since the natural substrates for the other enzymes may be substituted by one of several disaccharides.

A quantitative method for GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1----6fucosyltransferase activity with lectin affinity chromatography.

A quantitative method for the activity of GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1----6fucosyltransferase has been developed using a well-characterized substrate to which other fucosyltransferases fail to transfer and lentil lectin-Sepharose, which will bind this substrate only after fucosylation of the asparagine-linked N-acetylglucosamine. The enzyme was extracted from human skin fibroblasts and incubated with GDP-[14C]fucose and a specific substrate, asialo-agalactotransferrin glycopeptide. The product of the enzyme reaction, [14C]fucose alpha 1----6 to the asparagine-linked N-acetylglucosamine of the substrate, bound to lentil lectin-Sepharose and was eluted with 0.4 M methyl alpha-D mannopyranoside. The method was shown to be specific after characterization of the lentil lectin-bound glycopeptides by enzyme degradation and affinity chromatography. Quantitation of the method was shown by several parameters, including the linearity of product formed with respect to time, GDP-[14C]fucose concentration and enzyme concentration.

Fibrodysplasia ossificans progressiva presenting as osteomyelitis-like syndrome.

An 8-year-old boy presenting with fever, neck pain, and torticollis was eventually diagnosed as having fibrodysplasia ossificans progressiva (FOP). The initial bone scan was interpreted as being suggestive of vertebral osteomyelitis. Subsequent computerized tomographic studies (CT scan), however, demonstrated paravertebral soft-tissue calcification adjacent to, but not involving, intact cervical vertebrae. The acute process of FOP was thereby distinguished from that of osteomyelitis by use of CT scan.