My long-term research goal is to elucidate the molecular mechanisms regulating epithelial cell type-specific gene expression that are responsible for the diverse roles of epithelial cells in airway function and disease. The plasticity of cell differentiation is the major biological thrust of airway epithelium, maintaining mucociliary function under normal conditions and expressing squamous and keratinizing properties after injury. The nature of this plasticity is not known. It has been recognized that a similar """"""""squamous cell differentiation"""""""" is involved in the pre-neoplastic lesion of developing bronchogenic cancer. Our central hypothesi of this first award application is that the expression of SPR1 gene is essential for squamous cell differentiation in airway epithelium. The expression of small proline-rich protein (SPR1) gene largely occurs in squamou tissues and is associated with the suprabasal cell layer. In vivo, airway epithelium normally does not express any significant level of SPR1 gene products. However, a rapid induction of the expression of this gene occurs immediately after epithelial injury caused by various environmental pollutants Therefore, SPR1 can be used as a squamous cell biomarker for airway epithelium injury. We have demonstrated in our preliminary studies (in vitro and in vivo) that the 622 bp SPR1 promoter region contains the necessary information to direct high-level cell type-specific expression in squamous tissues and a basal-level expression in airway epithelium. We have also observed a strong correlation between tobacco smoke and TPA-induced squamous cell differentiatio and SPR1 gene expression in airway epithelium, mainly at the transcriptional level. Based on our preliminary data, we hypothesize that SPR1 promoter region contains regulatory elements for the binding of cell type-specific transcription factor(s) that are activated or induced during airway squamous cell differentiation. To test our hypothesis, we will use an in vitro cell culture system such as SPR1-expression airway epithelial cells and SPR1-nonexpressing fibroblast cells to characterize airway epithelial cell-specific molecular mechanisms. Specifically, we will focus on the mode of molecular mechanisms that are involved as follows: (1) cell type-specific basa expression of the human SPR1 gene in airway epithelium and (2) inducible expression of human SPR1 gene by TPA and tobacco smoke. We will use a combination of in vivo footprinting, deletion, site-directed mutagenesis, and gel mobility shift assays to identify cis-acting elements that are responsible for both the basal and inducible gene expression. Based on the gel mobility shift assay, we will use DNA affinity column and/or expression screening technique to isolate trans-acting protein factor(s) that are responsible for both basal and inducible gene expression. cDNA and immunological probes of these isolated trans-acting factor(s) will be characterized in vivo to elucidate tissue-specific and cell type-specific gene expression. This study will help us better understand the plasticity of airway epithelial cell differentiation and the response of airway epithelial cells to environmental insults.
| Reddy, Sekhar P M; Mossman, Brooke T (2002) Role and regulation of activator protein-1 in toxicant-induced responses of the lung. Am J Physiol Lung Cell Mol Physiol 283:L1161-78 |
| Kleeberger, S R; Reddy, S P; Zhang, L Y et al. (2001) Toll-like receptor 4 mediates ozone-induced murine lung hyperpermeability via inducible nitric oxide synthase. Am J Physiol Lung Cell Mol Physiol 280:L326-33 |
| Kleeberger, S R; Reddy, S; Zhang, L Y et al. (2000) Genetic susceptibility to ozone-induced lung hyperpermeability: role of toll-like receptor 4. Am J Respir Cell Mol Biol 22:620-7 |
