PERSPECTIVE
Airway Epithelium and Mucus
Intracellular Signaling Pathways for Gene Expression and Secretion

Kenneth B. Adler and Yuehua Li

The Department of Anatomy, Physiological Sciences and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina

It is the rare scientific paper dealing with any aspect of airway mucus that does not open with a statement about the contribution of excess mucus to the pathogenesis of airway obstruction, susceptibility to infection, or compromised defense in a myriad of inflammatory airway diseases, such as chronic bronchitis, asthma, bronchiectasis, or cystic fibrosis. Excess mucus in the airways can result from any of three different lesions, and in most cases various combinations of these: (1) enhanced production through overexpression of mucin (MUC) genes; (2) excess production secondary to mucus cell hyperplasia, hypertrophy, or even metaplasia; or (3) hypersecretion of formed and stored mucin by goblet cells or glands in the airways. In context of a perspective, it may be instructive to trace the historical pathways that have led to our present understanding of the mechanisms associated with mucus-related phenomena.

Clearly, the importance of studying production and secretion of mucus (or its glycoprotein component, mucin) was not lost on early researchers. In the 1960s and early 1970s, several groups looked at mucus production and secretion in the airways. However, lack of appropriate in vitro or in vivo model systems made these early studies mostly descriptive and limited, for the most part, to characterization of alcian blue/PAS-stained cells in different regions of the airways in health and disease (1). In the mid-1970s, with the introduction of organ culture techniques to study isolated rings or explants of bronchi or trachea from several species, it became possible to investigate mechanisms related to production and secretion of mucin (5, 6). Unfortunately, there were serious problems with explant cultures, not the least of which was quantification of produced or released mucin. The "state-of-the-art" at that time was either to measure carbohydrate components of secreted or retained mucin in the explants (such as sialic acid; fucose, or glucosamine [7]) or to incubate the explants with a radiolabeled sugar (such as tritiated glucosamine) for a time period allowing for incorporation of the label into the mucin glycoproteins, and then measure the released radiolabeled activity as a reflection of secreted mucin, or radioactivity within the tissue as a measure of mucin synthesis (8). For greater specificity, the homogenate or spent medium was either precipitated with trichloroacetic acid, sometimes with the addition of phosphotungstic acid, prior to counting of radioactivity. More accurate quantification was achieved by treatment of the homogenate or spent medium with enzymes to digest other contaminating sugar-containing proteins, such as hyaluronic acid or chondroitin sulfate, with hyaluronidase or chondroitinase ABC, respectively. Separation via column chromatography also improved detection, as the high molecular weight mucins would appear in the void volume (9). A second problem related to organ cultures was the large number of cell types present in the explants, confounding interpretation of effects of added agents on epithelium and making it difficult to attribute responses to any particular cell type.

There were some major advancements in the field of mucin research during the 1980s, both in the development of better cell culture techniques and in detection of intra- and extracellular (secreted) mucin. The first was a great improvement in our ability to culture cells from airway epithelium from several species. Prior to this time, it was difficult to culture airway epithelial cells so as to maintain differentiated characteristics in vitro, but the development of defined, serum-free medium, as well as improvements in the types of substrata beneath the cultured cells, gave researchers the ability to culture airway epithelial cells that looked and acted somewhat like their in vivo counterparts. Maintaining cells in a defined medium atop a collagen gel provided improved model systems, and in the latter part of the 1980s, the concept of air/liquid interface culture was first introduced. Starting with guinea pig tracheal epithelial cells (10, 11), it was discovered that cells grown on a collagen substrate, atop a permeant filter, with all medium placed beneath the cells and only a humidified air environment above, would result in well-differentiated epithelial cells essentially identical in structure and function to airway epithelium in situ. In quick succession, techniques for air/liquid interface culture of airway epithelium from rat (12), bovine (13), canine (14), primate (15), and eventually human (16) cells were developed. At present, culturing human airway epithelial cells in air/liquid interface provides a model system in which the epithelial cells are similar if not identical to human airway epithelium in vivo, and such cells now can be purchased commercially. With regard to detection of intracellular or secreted mucin, a major advance was the development of accurate and sensitive ELISA techniques to measure mucin, based on either polyclonal or monoclonal antibodies, usually against carbohydrate epitopes on the mucin molecule (19). With the identification and (at least partial) cloning of the different mucin genes (MUC1-4, 5AC, 5B, 6-9, and 11-13) from different species, it is now possible to quantitatively assess expression of these different mucin genes under normal conditions and in response to stimulation or repression. In addition, these tools, together with numerous advances in our abilities to monitor signal transduction pathways, have allowed for elucidation of intracellular signaling mechanisms associated with enhanced or decreased mucin gene expression or secretion.

These advances, however, are associated with a number of problems that are not unique to the mucus field, but probably permeate studies of airway epithelial structure and function in general. In reviewing the literature, one clearly can become confused by the apparent contradictions and conflicting data that are encountered routinely. Limiting this perspective to in vitro systems, much of this variability probably results from the different types of model systems of airway epithelium that have been utilized, differences in reagents and techniques used to measure mucin secretion and/or gene expression, and, of course, species specificity. The types of cell culture systems utilized can be divided into cell lines and primary cells. A large number of different airway epithelial cell lines have been developed and used, including (but certainly not limited to) NCI-H292, A-549, BEAS-2B, 16HBE14o, HBE1, and SPOC1 (Table 1). For the most part, although these cells express various mucin genes and release products that cross-react with antibodies to various mucin epitopes, these cells are not well-differentiated and may have different functional responses based on passage number and medium or substrate. More importantly, they are transformed and immortalized cells and therefore may differ in their responses and intracellular signaling pathways from epithelial cells in vivo. However, cell lines do provide the advantage of presenting essentially a homogeneous cellular population that can be easily manipulated and transfected, and they can be maintained in large numbers relatively easily and economically. Primary cells, on the other hand, provide a model system that is more differentiated and closer to the in vivo situation both structurally and functionally. As mentioned above, maintaining primary cells from a number of species using an air/liquid interface system seems to provide the best model for differentiated airway epithelium, but, unlike cell lines, these cultures contain a heterogeneous cellular population (e.g. ciliated, secretory, basal), show greater variability in a number of mucin-related and other responses, and are much harder to manipulate genetically or even molecularly. A good compromise appears to do studies with appropriate cell lines, but validate these findings in differentiated primary cells.

Given these caveats, there have been numerous studies demonstrating mucin gene enhancing and/or secretagogue effects of a variety of pathophysiological agents. Secretagogues include eicosanoids and lipid mediators (20), inflammatory mediators (21), environmental agents (22), bacterial-derived products (23), reactive oxygen and nitrogen species (24, 25), ATP and UTP (26, 27), and many other agents. Some substances that increase mucin gene expression include, for example, bacterial-derived products (28), cytokines (29), inflammatory mediators (30), and environmental pollutants (31). Interestingly, many of these agents appear to act through similar intracellular signaling pathways, and much effort over recent years has been directed toward identification of specific intracellular signal transduction pathways associated with altered production and/or secretion of mucin. This is not meant to be a comprehensive review of the area, but certainly some points that appear to be generally agreed upon are: (1) there are a large number of pathophysiological agents that can increase mucin secretion and/or mucin gene expression; (2) the major mucin genes in airway epithelium appear to be MUC5AC and perhaps MUC2; (3) mucin gene expression appears to involve one or more of the MAP kinase pathways, as well as intracellular redox state and activation of NF-kB (32, 33); and (4) mucin secretion appears to involve protein kinases and perhaps calcium and intracellular oxidants. Certainly, a case can be made for other pathways being important, and this is not meant to preclude these potentially important molecules.

In this issue of the American Journal of Respiratory Cell and Molecular Biology, a paper by Chen and coworkers from the University of California at Davis deals with intracellular molecular and signaling events related to stimulation of mucin secretion and gene expression by UTP in differentiated human bronchial epithelial cells in vitro. It is highly appropriate that a paper from Reen Wu's laboratory serve as the basis for this perspective, as Dr. Wu has made a number of substantial contributions to our understanding of airway epithelial function and mucin production and secretion. He developed a defined medium to allow for culture of primary airway epithelial cells in a paper that is oft-quoted as the basis for epithelial cell culture (34). He also did pioneering work on the importance of the substratum in maintaining the viability and differentiated characteristics of cultured airway epithelium (35), and was the driving force behind development of the air/liquid interface culture system described previously (10, 11). The paper in this issue demonstrates that Dr. Wu's laboratory continues to be in the forefront of research in this area. The important results in this paper relate to elucidation of the mechanisms by which nucleotide triphosphates act to enhance both mucin gene expression and mucin secretion in well-differentiated human airway epithelial cells. The fact that UTP enhances both secretion and gene expression, and appears to do so via different signaling pathways, represents a novel and potentially major finding. Of even greater interest, however, and in line with previous reports from this laboratory (36), it appears that the mucin gene MUC5B may be of great importance in both cultured cells and in a number of disease states in the airway. The fact that this group substantiated their findings in mice given UTP intratracheally in vivo is another outstanding feature of this paper.

This is not to say that there is not controversy regarding some of these findings. Certainly, there have been a number of reports in the literature implicating, for example, PKC in the pathway leading to enhanced expression of mucin genes in a number of other cell types (37, 38), and others have found no relation of the PLC pathway to mucin secretion (39). However, the paper points out some interesting directions for future studies in the area. Certainly, with frequent advances regarding cloning of the different MUC genes and development of better detection assays and reagents, it would appear that studies directed toward MUC5B, at both the gene and protein level, would be appropriate related to abnormal function. In addition, signaling pathways related to enhanced gene expression and secretion can be examined in much greater detail. For example, what are the phosphorylation targets of PKC in the secretory cells, and how do they act to enhance release of mucin granules? What role(s) do specific MAP kinases and their phosphorylation targets, and/or intracellular oxidants and oxidant-dependent transcription factors, play in enhancing mucin gene expression? With the availability of better reagents and technologies to address these and related questions, we certainly appear to be in a position to substantially increase our understanding of mucin production and secretion in the airways, leading ultimately to improved therapy and treatment for disease characterized by mucus hypersecretion and airway obstruction.

	Footnotes

Address correspondence to: Kenneth B. Adler, Ph.D., Department of Anatomy, Physiological Sciences and Radiology, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, NC 27606. E-mail: Kenneth_Adler{at}ncsu.edu

(Received in original form September 10, 2001).

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