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Elevated expression of gel-forming mucin (MUC) genes MUC5AC and MUC5B is a major pathological feature in various airway diseases. In this study, we show that phorbol 12-myristate 13-acetate (PMA) is a potent stimulator for MUC5B gene expression under air-liquid interface conditions in three airway epithelial cell systems: primary cultures of normal human bronchial epithelial cells, the immortalized normal bronchial epithelial cell line HBE1, and the human lung adenocarcinoma cell line A549. Stimulation was time- and dose-dependent, could be demonstrated by promoter-reporter gene transfection, and was sensitive to mithramycin A, suggesting the involvement of a specificity protein 1-based transcriptional mechanism in the stimulation. PMA-induced MUC5B message and promoter-reporter gene activity were specifically sensitive to inhibition of protein kinase C delta, which was further confirmed by the forced expression of dominant-negative mutant of protein kinase C delta. Regarding downstream transduction, PMA-induced MUC5B expression was sensitive to inhibitors and dominant-negative expression of signaling molecules involved in Ras/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase1-mediated c-Jun N-terminal kinase and p38 pathways. This contrasted with the inhibition of PMA-induced MUC5AC expression by inhibitors of the Ras/epidermal growth factor receptor/extracellular regulated kinase signaling pathway. These results demonstrate for the first time that PMA-stimulated MUC5AC and MUC5B expressions are regulated through distinctive epidermal growth factor receptor/extracellular regulated kinase-dependent and -independent signaling pathways.

Development of the high-density DNA microarray technique permits the analysis of thousands of genes simultaneously for their differential expression patterns in various biological processes. Through clustering analysis and pattern recognition, the significance of differentially expressed genes can be recognized and correlated with biological events that may take place inside the cell and tissue. With this notion in mind, high-density DNA microarray nylon membrane with colorimetry detection was used to profile the expression of smoke- and hydrogen peroxide-inducible genes in a human bronchial epithelial cell line, HBE1. On the basis of the time course of expression, at least three phases of change in gene expression could be recognized. The first phase is an immediate event in response to oxidant injury. This phase includes induction of the bcl-2 and mdm-2 genes, which are involved in the regulation of apoptosis, and the mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1) gene, that functions as a regulator of various mitogen-activated protein kinase activities. The second phase, usually 5 h later, includes the induction of various stress proteins and ubiquitin, which are important in providing the chaperone mechanism and the turnover of damaged macromolecules. The third phase, which is 5-10 h later, includes the induction of genes that are apparently involved in reducing oxidative stress by metabolizing reactive oxygen species. In this phase, enzymes associated with tissue and cell remodeling are also elevated. These results demonstrate a complex gene expression array by bronchial epithelial cells in response to the insult of oxidants that are relevant to environmental pollutants.

The effects of dexamethasone, a glucocorticoid analog, on interleukin 8 (IL-8) gene expression were studied in cultures of primary human tracheobronchial epithelial cells and an immortalized human bronchial epithelial cell line, HBE1 cells. Dexamethasone inhibited IL-8 mRNA and protein expression in a concentration- and time-dependent manner. The inhibition did not occur at the transcriptional level since both nuclear run-on activity and IL-8 promoter-reporter gene expression assay revealed no significant effect. Instead, there was a change in IL-8 mRNA stability in dexamethasone-treated cultures. Under actinomycin D treatment, IL-8 mRNA was quite stable in dexamethasone-depleted cultures, while in dexamethasone-pretreated cultures, IL-8 message was rapidly degraded within the first hour, then leveled off. When dexamethasone and actinomycin D were added simultaneously to dexamethasone-depleted cultures, IL-8 mRNA remained rather stable. When cycloheximide was used to inhibit new protein synthesis, dexamethasone-dependent inhibition was not observed. These results suggest that a posttranscriptional mechanism, which requires dexamethasone-dependent new protein synthesis, is involved in the regulation of IL-8 mRNA by dexamethasone in airway epithelial cells.

A rhesus monkey interleukin (IL)-8 cDNA clone with >94% homology to the human IL-8 gene was isolated by differential hybridization from a cDNA library of distal airways after ozone inhalation. In situ hybridization and immunohistochemistry showed increased IL-8 mRNA and protein levels in epithelial cells at 1 h but not at 24 h after inhalation of ozone. The appearance of IL-8 in airway epithelial cells correlated well with neutrophil influx into airway epithelia and lumens. Air-liquid interface cultures of tracheobronchial epithelial cells were exposed to ozone in vitro. We observed a transient increase in IL-8 secretion in culture medium immediately after ozone exposure and a dose-dependent increase in IL-8 secretion and mRNA production. In vitro neutrophil chemotaxis showed a parallel dose and time profile to epithelial cell secretion of IL-8. Treatment with anti-IL-8 neutralizing antibody blocked >80% of the neutrophil chemotaxis in vitro. These results suggest that IL-8 is a key chemokine in acute ozone-induced airway inflammation in primates.

The development of routine techniques for the isolation and in vitro maintenance of conducting airway epithelial cells in a differentiated state provides an ideal model to study the factors involved in the regulation of the expression of mucociliary differentiation. Several key factors and conditions have been identified. These factors and conditions include the use of biphasic culture technique to achieve mucociliary differentiation, the use of such stimulators as the thickness of collagen gel substratum, the calcium level, and vitamin A, and such inhibitors as the growth factors, epidermal growth factor and insulin, and steroid hormones, for mucous cell differentiation. Using the defined culture medium, the life cycle of the mucous cell population in vitro has been investigated. It was demonstrated that the majority of the mucous cell population in primary cultures is not involved in the replication of deoxyribonucleic acid (DNA). However, the mucous cell type is capable of self-renewal in culture, and this reproduction is vitamin A dependent. Furthermore, differentiation from nonmucous to mucous cell type can be demonstrated by adding back a positive regulator such as vitamin A to the "starved" culture. Cell kinetics data suggest that vitamin A-dependent mucous cell differentiation in culture is a DNA replication-independent process, and the process is inhibited by transforming growth factor-beta1.