This proposal is an interdisciplinary effort that focuses on defining the cellular and molecular mechanisms by which airway smooth muscle (ASM) orchestrates and perpetuates airway inflammation. The central hypothesis states that synthetic responses of ASM modulate airway inflammation and remodeling in asthma. Although many studies have identified mechanisms that regulate inflammatory cell trafficking and activation, few have asserted a role for myocytes in modulating airway inflammation. ASM synthetic responses are defined as secretion of cytokines, chemokines, growth factors and expression of cell adhesion molecules and matrix components. To test our central hypothesis, four Projects and three Core Units are proposed. Project 1 will characterize the molecular signaling pathways that regulate cytokine-induced synthetic responses in human ASM. Preliminary data demonstrate that selective inhibition of pathways activated by IL-1beta and TNF-alpha has differential effects on the modulation of synthetic functions by these cytokines. These findings support a central hypothesis that cytokines regulate ASM synthetic functions through the coordinated activation of diverse signaling events. Recently established techniques for the transfection and microinjection of human ASM cells will enable delineation of the precise molecular mechanisms by which cytokines activate these pathways. Project 2 will determine how cell-matrix receptors such as CD44 promote airway inflammation, remodeling and airway hyperresponsiveness (AHR) by altering ASM function. Allergen-induced AHR is markedly inhibited in mice treated with antibodies that inhibit CD44 and in CD44 null mice. However, IgE production and leukocyte recruitment were unaffected in these animals. These data support the hypothesis that local CD44-matrix interaction regulates AHR. Using genetically manipulated mice and segmental allergen challenge (SAC) models, the relative contribution of CD44 expression on hernatopoetic cells versus lung cells and the molecular basis by which CD44 modulates AHR and airway remodeling will be determined. Project 3 will define the role of G protein coupled receptor (GPCR) trafficking and signaling in modulating inflammatory responses of ASM. GPCRs play a critical role in regulating multiple ASM functions including contraction, relaxation, and synthetic function. The dynamic regulation of GPCRs results in their movement or trafficking within the cell. Recent studies support the hypothesis that such movement is controlled by multiple mechanisms and that GPCR trafficking plays an important role in regulating signaling. A comprehensive series of studies using ASM cultures and transgenic mouse models are proposed to determine the mechanisms by which GPCR localization and trafficking regulate ASM signaling and function. Project 4 will study the mechanisms by which epithelial-ASM or -fibroblast interactions foster airway remodeling and inflammation. Cultured epithelial cells from asthmatics manifest a pro-inflammatory, fibrogenic phenotype towards ASM cells and fibroblasts. These data support the hypothesis that epithelial cells from asthmatics secrete substance(s) that act on fibroblasts and ASM to promote the structural airway changes in asthma. Using SAC and a novel epithelial cell culture model, the factors that promote fibrogenesis and the signaling pathways they activate in mesenchymal cells will be determined. Three Core Units support four Projects. Core A will perform murine physiology, histology, cytology and will establish cell lines derived from wild type and transgenic mice. Core B will supply the Projects with physiological studies and clinical specimens from asthmatics and normals. Core C will provide administrative and fiscal support. Using hypothesis-driven molecular studies of genes, proteins, cells, tissues, animal models and asthmatic patients, this interdisciplinary program will also provide new insight into the pathogenesis and treatment of asthma.
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