ABSTRACT
The mucociliary epithelium plays an important role in regulating the homeostasis of conducting airways (1). An aberrant expression of mucociliary functions is frequently associated with various lung and airway diseases. The nature of the regulation of this aberrant expression is not known. In vitro culture of primary airway epithelial cells is an invaluable tool used to study, manipulate, and identify conditions that regulate cell differentiation. Our laboratory was first involved in the development of serum-free, hormone-supplemented medium to grow airway 225
epithelial cells from various species in culture, including those from humans (2). Subsequently and together with several other laboratories, we were able to demonstrate the importance of several factors contributing to airway epithelial cell differentiation in culture. These factors included the thickness of the collagen gel substratum (3), the calcium level in the medium (4), the vitamin A level, and an air/liquid interface culture condition (5-7). How these factors contribute to airway mucociliary differentiation in vitro is still unknown.We previously utilized differential and/or subtractive hybridization and differential display methods to identify those genes that are differentially expressed in the presence of vitamin A (8-10). These studies have established that several differentially expressed genes can be identified. For instance, the expression of a small proline-rich protein gene (SPRR1B), whose expression was shown to be elevated in cells and tissues with a squamous phenotype, was also enhanced in airway epithelial cell culture under vitamin A-depleted conditions (9). In addition, this elevation was consistently seen with other agents able to elevate various squamous cell functions (13), such as high calcium, UV light (11,12), and phorbol ester. Thus, these substances and vitamin A deprivation activate SPRR1B gene expression. We also demonstrated that, in addition to SPRR1B, several other genes were affected. One example was keratin gene expression, which was elevated in vitamin A-depleted culture condition (14). There should be several additional, not yet identified genes, whose expression is affected by these treatments. Since many genes are involved in each step of cell differentiation, an essential question for understanding cell differentiation is whether all these differentially expressed genes are coordinately regulated. The traditional approach using Northern blots to examine a small number of genes is insufficient to understand the differentiation process comprehensively. One of the research challenges in the postgenomic period is to have a high-throughput system to identify all genes involved and the pattern of the gene expression associated with the process. The recent development of high-density DNA microarray membranes (15) and glass slides (16,17) is quite appropriate for this challenge. With microarrays, the expression level of thousands of genes can be investigated simultaneously using a small area, which can be easily handled for hybridization, quantitation, and analysis. Thus, thousands of genes can be analyzed together, and genes that are differentially expressed can be identified.The purpose of this chapter is to utilize the newly developed technology of microarray membranes to analyze genes whose expression is associated with mucociliary differentiation of human airway epithelial cells in vitro. Two types of nylon membranes were used. One contains 884 sequence-verified expression sequence tag (EST) clones, the other contains 576 uni-EST clones. Data obtained from these membranes were further characterized by Northern blot hybridization.
