Asthma is a complex disease with increasing incidence causing significant morbidity and mortality. Interleukin-4 (IL-4) and interleukin- 13 (IL- 13) are functionally and structurally related cytokines released by T lymphocytes that are implicated as central mediators of asthma. Functional similarities are attributed to the shared receptor subunit, IL-4Roc. Despite many redundancies, substantial functional differences exist between IL-4 and IL- 13 in vivo and in vitro, implicating a greater role for IL- 13 in airway remodeling and airway hyperreactivity, which are important features in the pathogenesis of asthma. Furthermore, our preliminary data demonstrate important differences in transcriptional effects of IL-4 and IL-13 in bronchial smooth muscle cells (BSMC) and normal human lung fibroblasts (NHLF). The mechanism(s) for these differences are poorly understood. In the current application, I propose to study the mechanism(s) underlying these transcriptional differences and to identify the differentially regulated genes which may be of functional significance. In the first aim, I expect to identify numerous additional genes differentially regulated by IL-4andIL-13. This will be accomplished by gene expression profiling of airway cells studied at multiple times after stimulation with a range of concentrations of IL-4 or IL- 13 using oligonucleotide arrays containing probes for 14,000 genes. In the second and third aims, I propose to identify the specific mechanism(s) by which IL-4 and IL-13 regulate differential transcriptional effects by systematically examining components of IL4 and IL-1 3 signaling pathways including receptors and signaling mediators. These studies will utilize immunoprecipitation, immunoblotting, reverse transcriptase PCR, and competitive receptor binding assays. Finally, to examine the direct relevance of these differentially regulated genes in vivo, I will examine STAT-6 dependence of these genes in STAT-6 knockout mice with tissue-specific reconstitution of STAT-6 in air-way cells. This approach will allow me to directly assess the importance of a central component of known IL-4 and IL- 13 signaling pathways in vivo. Understanding mechanism(s) underlying IL-4 and IL- I 3's differential contributions to asthma pathogenesis as well as identifying key genes which may be of functional relevance may ultimately lead to better design of novel treatments of asthma. Together with proposed coursework, journal clubs and seminars, and with regular meetings with my mentor and advisory committee, this training program should allow me to develop the skills and experience necessary to become a successful independent investigator and University faculty member.
