Focalized proteolysis: spatial and temporal regulation of extracellular matrix degradation at the cell surface.

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.

Morphological studies of stimulated adrenergic axon varicosities in the mouse vas deferens.

The postganglionic axons of sympathetic neurons innervating the mouse vas deferens were stimulated transmurally in vitro by passing square pulses between two platinum electrodes. The ultrastructural appearance of the adrenergic nerve terminals was compared to samples fixed immediately after 30 min of stimulation and in samples allowed to recover for 2 h before fixation. The contralateral vasa deferentia served as controls, and these were incubated in Krebs solution for the same period as stimulated muscles. For each of four experiments, the mean number of large and small dense-core vesicles per square micrometer was calculated, as were the mean area and perimeter of the axon varicosities in each group. It was found that the number of small vesicles per square micrometer decreased by 60% during the stimulation period, but returned almost to control levels 2 h later. Large vesicles did not change in number during the stimulation or recovery periods. The proportion of vesicles containing cores was also determined for each group and found to decline just after stimulation in the small vesicle population, but to remain constant in the large vesicle population. The core depletion was partly reversed after 2 h. The vesicle recovery process was studied by use of the extracellular tracer horseradish peroxidase (HRP). When HRP was present in the extracellular space during stimulation, large numbers of vesicles contained the marker after recovery from stimulation. Thus, it is proposed that adrenergic axon varicosities recycle vesicle membrane through the plasma membrane in a manner similar to that already described for cholinergic nerve terminals.

Localization of Na pumps in the tracheal epithelium of the dog.

Binding of [3H]ouabain by the dog's tracheal epithelium shows a nonspecific component depending linearly on ouabain concentration, and a specific saturable component with a Km of 10(-7) M. Control experiments showed that the tracer taken up was not trapped within the extracellular space nor bound to tissue collagen. Inhibition of the saturable uptake by high K, metabolic inhibition, low Na, and low temperature indicated that binding was to Na/K ATPase. One-sided exposures of tissue sheets to tracer showed that the submucosal side took up 10 X as much tracer as the luminal. Autoradiography localized tracer uptake under all conditions to the cells' basolateral membranes.

Neutrophil elastase and cathepsin G stimulate secretion from cultured bovine airway gland serous cells.

To investigate the hypothesis that neutrophil proteases stimulate airway gland secretion, we studied the effect of human cathepsin G and elastase on secretion of 35S-labeled macromolecules from cultured bovine airway gland serous cells. Both proteases stimulated secretion in a concentration-dependent fashion with a threshold of greater than or equal to 10(-10) M. Elastase was more potent than cathepsin G, causing a maximal secretory response of 1,810 +/- 60% over baseline at 10(-8) M. The maximal response to cathepsin G (1,810 +/- 70% over baseline at 10(-7) M) was similar to the maximal response to elastase. These responses were greater than 10-fold larger than the response to other agonists such as histamine. Protease-induced secretion was noncytotoxic and required catalytically active enzymes. The predominant sulfated macromolecule released by proteases was chondroitin sulfate proteoglycan. Immunocytochemical staining demonstrated chondroitin sulfate in cytoplasmic granules and decreased granular staining after stimulation of cells with elastase. The neutrophil proteases also degraded the proteoglycan released from serous cells. Cathepsin G and elastase in supernatant obtained by degranulation of human peripheral neutrophils also caused a secretory response. Thus, neutrophil proteases stimulate airway gland serous cell secretion of chondroitin sulfate proteoglycan and degrade the secreted product. These findings suggest a potential role for neutrophil proteases in the pathogenesis of increased and abnormal submucosal gland secretions in diseases associated with inflammation and neutrophil infiltration of the airways.

Human bronchus and intestine express the same mucin gene.

The amino acid and sugar composition of mucins from various organs is similar but not identical. This could arise by one or more of the following: organ-specific processing of a single core protein, organ-specific splicing of a single mucin mRNA, or organ-specific expression of various mucin genes. To begin to investigate the source of this variability, we examined (a) immunological cross-reactivity and (b) cDNA cross-hybridization, among several mucin-secreting organs of the human body. Peptide-directed antibodies raised against both nondeglycosylated (LS) and deglycosylated (HFB) intestinal mucin strongly stained mucous cells in the bronchial epithelium and submucosal glands, indicating homology between mucins of the bronchus and intestine at the peptide level. By screening a bronchus cDNA library with an intestinal mucin cDNA, SMUC-41, we isolated a bronchus mucin cDNA, HAM-1. This cDNA is 96% homologous to the first repeat of SMUC-41. HAM-1 hybridized to restriction fragments of human genomic DNA identical to those hybridizing to SMUC-41 on Southern blots. SMUC-41 also hybridized to polydisperse transcripts in the bronchus, cervix, gall bladder, and mammary gland, indicating mucin homology among all these organs at the RNA level. We conclude that the bronchus and intestine express a common mucin gene, which is likely co-expressed by at least several other mucin-secreting organs.

Allergen-induced IL-9 directly stimulates mucin transcription in respiratory epithelial cells.

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.

Characterization of quantitative mucin variants from a human colon cancer cell line.

Colonic mucins are high molecular weight glycoproteins produced by goblet cells of colonic epithelium. Some studies have indicated that patients with colonic cancers that produce high amounts of mucin have a poorer prognosis than patients whose tumors produce low amounts of mucin. At present, however, the role of mucin in affecting the behavior of colon cancer cells is not well understood. To further elucidate the relationship between cellular mucin content and the growth characteristics and morphology of tumor cells, we utilized a replica plating technique and immunoscreening method to identify and purify variant clones of the human colon cancer cell line LS174T that produce high and low levels of mucin. This procedure enabled us to isolate two high mucin-containing variants (HM3 and HM7) and one low mucin-containing variant (LM12). These variants exhibited different morphology. Both high mucin variants tended to form cell aggregates and suspended cells with adjoining mucoid threads. The low mucin variant formed spread monolayers on the substratum with the formation of cell processes. Metabolic labeling using [3H]glucosamine demonstrated that high mucin variants synthesized 2-fold more mucin in the cell layer and secreted 3-fold more mucin into the culture medium than the low mucin variant. The colony-forming efficiency in semisolid agar for these variants positively correlated with their mucin content. High mucin variant cells when injected into athymic nude mice formed tumors 2-fold larger than those of the parental cells while the low mucin variant formed tumors only one-half as large as those of the parental cell line. These mucin variants should provide a useful model for understanding the biological behavior of mucinous colon cancer cells in vivo and in vitro.

Mucin gene (MUC 2 and MUC 5AC) upregulation by Gram-positive and Gram-negative bacteria.

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.

Identification of decorin proteoglycan in bovine tracheal serous cells in culture and localization of decorin mRNA in situ.

Bovine tracheal submucosal gland serous cells in culture synthesize and secrete proteoglycans and not mucin glycoconjugates. We are interested in the characterization and role of these proteoglycans in airway secretions. The major [35S]methionine-labeled proteoglycan present is identified as the small chondroitin/dermatan sulfate proteoglycan decorin (PG II. PG40). Consistent with its identity as decorin this proteoglycan showed average apparent molecular weights of 75,000 to 130,000 with a core protein of an average, M(r) of about 40,000 and with glycosaminoglycan chains sensitive to chondroitinase ABC lyase of an average M(r) of about 25,000. These data were obtained from gel chromatographic and SDS-PAGE analyses. Northern blot analysis and partial amino acid sequencing of the purified protein further confirmed its identity as decorin. In situ hybridization studies using a decorin riboprobe revealed no expression of decorin in the surface epithelium and only low levels of expression in submucosal gland epithelial cells of bovine tracheal tissue. However, high levels of expression were localized to cells which are peripheral to tracheal submucosal gland epithelial cells and which contact with the extracellular matrix.

Sp1 protein contributes to airway-specific rat MUC 2 mucin gene transcription.

We have shown increases in the abundance of airway mucin mRNA during the pathogenesis of chronic obstructive pulmonary disease in rat models (Jany et al., 1991) and now seek to determine the underlying mechanisms. As transcriptional modulation may be involved, we provide here a functional analysis of the 5' flanking region of a rat mucin gene (MUC 2). Using deletion mutants to bp -859, we constructed expression cassettes in CAT vectors and transfected them into two MUC 2-expressing cell lines, SPOC 1, a rat airway epithelial cell line and IEC-6, a rat intestinal epithelial cell line, and into one MUC 2 non-expressing cell line, FR, a rat skin fibroblast cell line. Results indicated that nucleotides -59 to -40 mediated high level expression in SPOC 1, but not in the other cells. Used as a probe in gel shift assays, fragment -59/-40 formed complexes of differing mobilities when incubated with nuclear protein extracts from the three cell types. Mutation of the putative Sp1 binding site in the probe sequence interfered with protein binding in all three cell types, but anti-Sp1 antibody supershifted a band formed only by airway cell extracts. A model of airway cell-specific MUC 2 transcription is proposed.

Tumor-derived EMMPRIN (extracellular matrix metalloproteinase inducer) stimulates collagenase transcription through MAPK p38.

EMMPRIN (extracellular matrix metalloproteinase inducer) stimulates fibroblast metalloproteinases (MMP) 1, 2 and 3 (Kataoka et al. (1993) Cancer Res. 53, 3154-3158). Here we focus on MMP-1, showing that in lung tumors, MMP-1's cognate mRNA is strongly expressed in stromal fibroblasts adjacent to EMMPRIN-expressing tumor cells. In vitro, EMMPRIN upregulates MMP-1 mRNA expression in a concentration-dependent manner, with a peak accumulation at 24 h. The response is genistein-sensitive, suggesting it is dependent on tyrosine kinase activity. Analysis of tyrosine phosphorylation-dependent MAP kinases ERK 1/2, SAPK/JNK, and p38 showed that the activity of p38 but not that of the other 2 kinases was elevated in response to EMMPRIN. That p38 activity was required for EMMPRIN stimulation of MMP-1 was evident from results showing that the p38 inhibitor SB203580 blocked this response. This is the first available information regarding the mechanism by which tumor-associated molecules upregulate MMP synthesis in stromal fibroblasts.

The architecture of nerves and ganglia of the ferret trachea as revealed by acetylcholinesterase histochemistry.

The goal of this study was to determine the architecture of the nerves and ganglia of the ferret trachea. Tracheas from four newborn ferrets and three adult ferrets were stained histochemically for acetylcholinesterase activity and analyzed in their entirety as whole mounts. The architecture consisted of one or two longitudinal nerve trunks overlying the posterior surface of the trachealis muscle, a dense plexus of nerves superficial to the trachealis muscle that interconnected these longitudinal nerve trunks, and, on the anterior surface, a plexus superficial to the submucosal glands and located between the cartilaginous rings. In addition, deep neural plexuses were associated with the trachealis muscle and with the submucosal glands. Ganglion cell bodies along the longitudinal nerve trunks were large (mean diameter +/- S.E. = 34.3 +/- 0.3 microns), were usually attached to the nerve trunk by a stalk, and were loosely clustered in groups of as many 38 cell bodies. By contrast, those cell bodies of the superficial muscle and gland plexuses were significantly smaller (mean diameter +/- S.E. = 24.2 +/- 0.3 microns), were never attached by a stalk, and were tightly clustered in ganglia of one to four cell bodies. We conclude that nerves and ganglia of the ferret trachea constitute one or two longitudinal nerve trunks containing ganglia with large cell bodies, two superficial nerve plexuses containing ganglia with small cell bodies overlying the smooth muscle and submucosal glands, respectively, and two deep nerve plexuses providing the terminal innervation to the muscle and glands.

Altered mucin expression is a field change that accompanies mucinous (colloid) breast carcinoma histogenesis.

Mucinous carcinomas of the breast, so-called colloid carcinomas, exhibit better prognoses than their nonmucinous breast counterparts. This biological difference exhibited by mucinous breast carcinomas prompted us to examine the relationship of mucin expression to colloid carcinoma histogenesis. We studied 50 colloid carcinomas, 50 noncolloid cancers, and 50 normal breasts by hematoxylin-eosin (H&E) and Alcian blue staining, mucin immunohistochemistry, in situ hybridization with a battery of MUC riboprobes, and ancillary digital image analysis. We observed luminal mucin in normal ducts in 80% of colloid carcinomas compared with 10% of noncolloid carcinomas and 6% of normal breasts (P < .01). In the cases of colloid carcinoma that showed mucin-filled ducts, luminal mucin was observed in 40% of the normal ducts and acini, 40% to 75% of the ducts involved by hyperplasia, atypical ductal hyperplasia (ADH) and ductal carcinoma in situ (DCIS), respectively, and in 50% of the co-incidental areas of cysts (mucoceles), adenosis, fibroadenoma, and intraductal papilloma (P < .01). Immunohistochemistry showed that colloid carcinomas showed strong MUC2 cytoplasmic immunoreactivity and decreased MUC1 immunoreactivity compared with noncolloid carcinomas. In situ hybridization studies indicated fivefold increased MUC2 signals and twofold increased MUC5 signals within adjacent and remote normal epithelium in only the colloid carcinoma cases (P < .01; P < .05). In these cases of colloid carcinoma, these increased MUC2 and MUC5 signals were also observed in areas of hyperplasia, ADH, DCIS, and invasive carcinoma. In contrast, the noncolloid carcinomas showed fivefold increased MUC1 signals but no increases in MUC2 or MUC5. In mixed colloid/noncolloid carcinomas, the colloid areas had identical mucin expression patterns as the pure colloid carcinomas, but there was a loss of MUC2 and MUC5 expression and a gain of MUC1 expression in the noncolloid areas that was therefore identical to the pattern observed in pure noncolloid carcinoma. In this study, we conclude that the altered expression of mucin so characteristic of colloid carcinoma is also a field change present in adjacent and remote normal breast epithelium.

In vivo activity of tracheal parasympathetic ganglion cells innervating tracheal smooth muscle.

In vivo intracellular recording and intrasomal injection of Lucifer yellow revealed two populations of postganglionic parasympathetic neurons in the tracheal ganglia of cats. One consisted of large cells that had an inspiratory rhythm, had a significant post-spike afterhyperpolarization, and projected to the tracheal smooth muscle. The second consisted of small cells that fired with an expiratory rhythm, had no significant afterhyperpolarization, and projected to the intercartilaginous spaces.

Muscarinic receptors in lung and trachea: autoradiographic localization using [3H]quinuclidinyl benzilate.

[3H]Quinuclidinyl benzilate binding to slide-mounted frozen sections of ferret lung was of high affinity (KD 63 +/- 14 pM, mean +/- S.E., n = 4), and characteristic of interaction with muscarinic cholinergic receptors. Light microscopic autoradiography showed muscarinic receptors to be localized predominantly to smooth muscle of trachea and intrapulmonary cartilaginous airways, and to submucosal glands. There was much less labelling of bronchiolar smooth muscle, airway epithelium and vascular smooth muscle and no labelling of alveoli. This distribution of receptors parallels that of cholinergic innervation.

Pulmonary alpha-adrenoceptors: autoradiographic localization using [3H]prazosin.

We determined the distribution of pulmonary alpha-adrenoceptors by autoradiographic localisation of [3H]prazosin binding to frozen sections of ferret lung. Specific binding of [3H]prazosin to lung sections was saturable and of high affinity (KD = 0.44 +/- 0.55 nM; mean +/- S.E., n = 5), with a specificity indicating binding to alpha 1-receptors. Autoradiographic showed that alpha 1-receptors were present in highest density in vascular smooth muscle (small vessels greater than large vessels), and were also present in airway submucosal glands and epithelium. There was also scanty labelling of alveolar walls which may be to contractile interstitial cells (Kapanci cells). Although smooth muscle of bronchi showed little labelling, surprisingly that of bronchioles was heavily labelled. The high density of alpha-receptors in small airways may be relevant to asthma in which alpha-adrenergic responses are activated. This method offers a means by which autonomic receptors of small airways may be investigated without the confusing contribution of other contractile elements and larger airways.

Transmission in airway ganglia of ferrets: inhibition by norepinephrine.

We examined the possibility that norepinephrine inhibits transmission in parasympathetic ganglia of the ferret trachea. We impaled ganglion cells on recording microelectrodes and evoked postsynaptic action potentials by stimulating fiber tracts entering the ganglion. When norepinephrine was added to the recording bath, the action potentials were blocked. Phentolamine reversed this block. These results indicate that, by activating alpha-receptors, norepinephrine inhibits transmission in airway ganglia.

Regulation of airway secretory cells.

Inhaled particles are cleared from the airways by ciliary transport of a discontinuous mucous layer. The effectiveness of this process depends on the viscoelastic properties of the secretion, which in turn depend on the composition and interaction of the glycoprotein constituents. In human airways, two major cell types in the surface epithelium (goblet and ciliated) and two in the submucosal glands (serous and mucous) are known to contain mucin- and/or serum-type glycoproteins. This article summarizes current knowledge regarding the secretable products of each of these cell types and the conditions under which they are released into the airway lumen.

Tracheal gland mucous cells stimulated in vitro with adrenergic and cholinergic drugs.

To determine the responsiveness of tracheal mucous cells to adrenergic and cholinergic stimulation, we analyzed changes in their structure induced by neurotransmitter-like agonists. Ferret tracheal rings were exposed for 30 min in vitro to one of the following: phenylephrine, isoproterenol, or bethanechol (all at 10(-5) M), in the presence of absence of appropriate antagonists. Electron microscopy and morphometric analysis revealed that the volume density of mucous cells (Vvmc, i.e. the space occupied by mucous cells in the submucosa) significantly decreased, and the surface density of mucous cell apical membrane (Svam) increased in response to isoproterenol and bethanechol but not to phenylephrine. In metabolic labeling experiments, the morphological changes were accompanied by secretagogue-evoked release of 35S-labeled macromolecules. Taken together, these data suggest that tracheal mucous cells secrete 35S-labeled macromolecules in response to beta-adrenergic and muscarinic agonists by an exocytotic process that involves a reduction in cell size.

Modification of mucin gene expression in the airways of rats exposed to sulfur dioxide.

The molecular mechanisms mediating mucous cell metaplasia and hypersecretion in the respiratory tract are unknown. Previous work suggests that mucous metaplasia requires the induction of mucin gene expression. We are investigating this possibility by monitoring steady state levels of mucin mRNA in a model of mucous cell metaplasia induced by SO2 exposure. Male Sprague Dawley rats were exposed to 400 ppm SO2 gas in air for 3 h per day, 5 days per week for 0,1,2, or 3 weeks. Sham controls were exposed to air under similar conditions. After 3 weeks, morphological changes were apparent in the epithelium of SO2 exposed rats at all levels from the trachea to the distal airways. The epithelial thickness increased, as well as the number and size of glands in the trachea. Epithelial mucous (goblet) cells increased from 0 to 4.5 per mm in the trachea, 0.2 to 6.2 per mm in the main stem bronchi, and 0.2 to 22.7 per mm in the distal airways (mean values obtained for 3-6 tissue blocks per airway level per condition). In parallel experiments, we used SMUC41, a 950 bp human intestinal cDNA to isolate a human airway cDNA, HAM-1 from a cDNA library constructed in bacteriophage from human bronchial poly A+RNA. HAM-1 is a 90 bp cDNA encoding a threonine- and proline-rich peptide with 96% homology to the human intestinal cDNA SMUC-41. Next we probed total and poly A+ airway RNA from rats in each exposure condition with SMUC-41 or HAM-1. Blots were then stripped and reprobed with cDNA encoding beta actin. Densitometry values normalized for the amount of RNA loaded per lane (as determined by actin hybridization intensity) showed that mucin mRNA increased 8-9 fold as a function of SO2 exposure. This is consistent with the possibility that mucin gene transcription is induced by SO2 exposure, and may represent a primary event in the development of mucous metaplasia and hypersecretion.